Functionalization of gold nanoparticles for cell transfection

Abstract This report focuses on gold nanoparticle functionalization and checks their capability to transfect cells with a DNA plasmid, so the possibilities of the gold nanoparticles as vehicles for gene therapy can be explored. The nanoparticles were covered with a short polymer as stabilizer and, by the formation of an amide bound, different molecules of interest were conjugated to interact with a plasmid. Cell viability in presence of those functionalized nanoparticles was checked, and, finally, a transfection assay was performed. Transfected cells produced EGFP (Enhanced Green Fluorescent Protein), so they could be identified with fluorescence microscopy. Though some cells were transfected, the amount is really low, so more optimizations of the functionalization would be required to improve the process efficiency. Resumen Con intención de explorar las posibilidades de las nanopartículas de oro como vehículos para terapia génica, este trabajo se centra en su funcionalización y en comprobar su capacidad para transfectar células con un plásmido de ADN. En la funcionalización se han recubierto las nanopartículas con un polímero de cadena corta como estabilizante, y a éste, mediante formación de grupos amina, se le han conjugado distintas moléculas de interés que puedan interactuar con un plásmido. También se ha comprobado la viabilidad de células Vero en presencia de las nanopartículas funcionalizadas, y por último se ha realizado un ensayo de transfección. Las células transfectadas producían EGFP (Enhanced Green Fluorescent Protein) para ser identificadas por microscopía de fluorescencia. Aunque algunas células sí que se transfectaron en el proceso, la cantidad es muy baja, por lo que se requerirían optimizaciones de la funcionalización para incrementar la eficiencia del proceso

Synthesis and functionalization of biocompatible Tb:CePO4 nanophosphors with spindle-like shape

Monoclinic Tb:CePO4 nanophosphors with a spindle-like morphology and tailored size (in the nanometer and micrometer range) have been prepared through a very simple procedure, which consists of aging, at low temperature (120 C), ethylene glycol solutions containing only cerium and terbium acetylacetonates and phosphoric acid, not requiring the addition of surfactants or capping agents. The influence of the heating mode (conventional convection oven or microwave oven) and the Tb doping level on the luminescent, structural and morphological features of the precipitated nanoparticles have also been analyzed. This study showed that microwave-assisted heating resulted in an important beneficial effect on the luminescent properties of these nanophosphors. Finally, a procedure for the functionalization of the Tb:CePO4 nanoparticles with asparticdextran is also reported. The functionalized nanospindles presented negligible toxicity for Verocells, which along with theirs excellent luminescent properties, make them suitable for biomedical applications.Peer Reviewe

15 years on siRNA delivery: Beyond the State-of-the-Art on inorganic nanoparticles for RNAi therapeutics

RNAi has always captivated scientists due to its tremendous power to modulate the phenotype of living organisms. This natural and powerful biological mechanism can now be harnessed to downregulate specific gene expression in diseased cells, opening up endless opportunities. Since most of the conventional siRNA delivery methods are limited by a narrow therapeutic index and significant side and off-target effects, we are now in the dawn of a new age in gene therapy driven by nanotechnology vehicles for RNAi therapeutics. Here, we outlook the >do's and dont's> of the inorganic RNAi nanomaterials developed in the last 15 years and the different strategies employed are compared and scrutinized, offering important suggestions for the next 15.This work has been funded by ERANET-NANOSCIERA NANOTRUCK project. JC acknowledges Marie Curie International Outgoing Fellowship (FP7-PEOPLE-2013-IOF, project no. 626386). JMF acknowledges SAF2014-54763-C2-2-R project (Spanish Government), European Regional and Social Development Funds, Aragón Autonomous Government (DGA) through Research Groups and ERC-Starting Grant 239931-NANOPUZZLE.Peer Reviewe

Gold nanoparticle-siRNA mediated oncogene knockdown at RNA and protein level, with associated gene effects

[Aims]: RNAi is a powerful tool for gene silencing that can be used to reduce undesirable overexpression of oncogenes as a novel form of cancer treatment. However, when using RNAi as a therapeutic tool there is potential for associated gene effects. This study aimed to utilize gold nanoparticles to deliver siRNA into HeLa cells. [Results]: Knockdown of the c-myc oncogene by RNAi, at the RNA, protein and cell proliferation level was achieved, while also identifying associated gene responses. [Discussion]: The gold nanoparticles used in this study present an excellent delivery platform for siRNA, but do note associated gene changes. [Conclusion]: The study highlights the need to more widely assess the cell physiological response to RNAi treatment, rather than focus on the immediate RNA levels.The authors acknowledge EPSRC for funding.Peer Reviewe

Highly sensitive ratiometric quantification of cyanide in water with gold nanoparticles via Resonance Rayleigh Scattering

A highly sensitive and selective ratiometric sensor for the quantification of cyanide (CN) in aqueous samples has been developed using spherical gold nanoparticles (AuNPs) stabilized by polysorbate 40 (PS-40). Three different AuNP sizes (14, 40 and 80 nm mean diameters) were used to evaluate the response of the sensor using both colorimetric and Resonance Rayleigh Scattering (RRS) detection schemes. The best results were obtained for the sensor using 40 nm AuNPs, for which the limits of detection (LODs) were found to be 100 nmol L in a benchtop instrument and 500 nmol L by the naked eye, values well below the maximum acceptable level for drinking water (1.9 µmol L) set by the World Health Organization (WHO). The practical use of the 40 nm-AuNPs RRS sensor was demonstrated with the determination of CN in drinking and fresh waters. Finally, the sensor was successfully implemented in a compact portable device consisting of two light-emitting diodes (LEDs) and a miniature spectrometer, turning this sensor into a very potent tool for its application as a quick routine field-deployable analytical method.The authors gratefully acknowledge financial support from Programa Nacional de Innovación para la Competitividad y Productividad, Innóvate-Perú (Grant No. 119-PNICP-PIAP-2015), Dirección de Gestión de la Investigación at PUCP (Grant No. DGI-2015-178), the Chemistry Section at PUCP and Fondo Social de la DGA (grupos DGA).Peer Reviewe

Significance of the balance between intracellular glutathione and polyethylene glycol for successful release of small interfering RNA from gold nanoparticles

The therapeutic promise of small interfering RNAs (siRNAs) for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol (PEG) ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels (cells in S-phase) by in-cell westerns and BrdU incorporation, respectively.Peer Reviewe

Coating an adenovirus with functionalized gold nanoparticles favors uptake, intracellular trafficking and anti-cancer therapeutic efficacy

Adenoviral (Ad) vectors have proven to be important tools for gene and cell therapy, although some issues still need to be addressed, such as undesired interactions with blood components and off-target sequestration that ultimately hamper efficacy. In the past years, several organic and inorganic materials have been developed to reduce immunogenicity and improve biodistribution of Ad vectors. Here we investigated the influence of the functionalization of 14 nm PEGylated gold nanoparticles (AuNPs) with quaternary ammonium groups and an arginine-glycine-aspartic acid (RGD)-motif on the uptake and biodistribution of Ad vectors. We report the formation of Ad@AuNPs complexes that promote cell attachment and uptake, independently of the presence of the coxsackievirus and adenovirus receptor (CAR) and αvβ3 and αvβ5 integrins, significantly improving transduction without limiting Ad bioactivity. Besides, the presence of the RGD peptide favors tumor targeting and decreases Ad sequestration in the liver. Additionally, tumor delivery of a coated Ad vector expressing the human sodium iodide symporter (hNIS) by mesenchymal stem cells induces increased accumulation of radioactive iodine (131I) and tumor volume reduction compared to naked Ad-hNIS, highlighting the promising potential of our coating formulation in cancer gene therapy. Statement of significance: Modification of adenoviral vectors with lipids and polymers can reduce interactions with blood components and increase tumor accumulation; however, increased toxicity and reduced transduction efficiency were indicated. Coating with gold nanoparticles has proven to be a successful strategy for increasing the efficiency of transduction of receptor-defective cell lines. Here we explore the contribution of cell surface receptors on the mechanisms of entry of Ad vectors coated with gold nanoparticles in cell lines with varying degrees of resistance to infection. The enhancement of the anti-tumoral effect shown in this work provides new evidence for the potential of our formulation.This research was supported by Instituto de Salud Carlos III (ISCIII) (PI19/01007) and by Fondo Europeo de Desarrollo Regional (Feder) “Una manera de hacer Europa”. We also thank CIBER-BBN, an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III (ISCIII) with the assistance of the European Regional Development Fund. This study was also partially funded by the Aragon Government (Ph.D. Grant No.r B054/12) and cofounded by Aragon/FEDER 2014–2020 “Building Europe from Aragon”