Polymer functionalized gold nanoparticles as non-viral gene delivery reagents.

Background: In this study we investigated the ability of PEG functionalized gold nanoparticles as non-viral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. Methods: Positively charged gold nanoparticles were synthesized using PEI as reducing and stabilizer agent and its cytotoxicity reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). The vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was indirectly evaluated using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. Results: An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA together with a reduced size and an elevated electrokinetic potential are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40kbp (plasmid size close to the limit of the DNA carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and HEK 293T) and suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. Conclusions: Non-viral vectors based on gold nanoparticles covalently coupled with polyethylene glycol (PEG) and Polyethylenimine (PEI) can be used as efficient transfection reagents showing expression levels same or greater than the ones obtained with commercially available lipoplexes.pre-print3905 K

The effect of hollow gold nanoparticles on stem cell migration. Potencial application in tissue regeneration.

Every year trauma together with bone, joints and cartilage-associated diseases usually involve structural damage, resulting in a severe pain and disability for millions of people worldwide[1]. In regenerative medicine, cellular, tissue and organ-based approaches are developed to restore biological functions that have been lost[2],[3]. Therefore, tissue repair and regenerative medicine have attracted the interest of the scientific community, providing promising results in preclinical models and clinical pilot studies.pre-print3341 K

Selective delivery of photothermal nanoparticles to tumors using mesenchymal stem cells as Trojan horses

The main challenge of cancer treatment is to avoid or minimize systemic side effects in off-target tissues. Mesenchymal stem cells (MSCs) can be used as therapeutical carriers because of their ability to migrate and incorporate into inflammation areas including tumors. Here, this homing ability is exploited by carrying therapeutic nanoparticles (Hollow Gold Nanoparticles (HGNs)) following a “Trojan-horse” strategy. Amongst the different nanoparticles to be employed, HGNs have the capacity to resonate in the near infrared region when irradiated by an appropriated laser (808 nm). By transforming this absorbed energy into heat, they are capable to produce locally induced hyperthermia. At this wavelength healthy tissues have a minimal light absorption being the effect restricted to the tissues containing HGNs. By placing HGNs inside MSCs, the recognition, excretion and immune response are minimized. We demonstrate that MSCs internalize HGNs and reach the tumors still containing HGNs. After laser treatment this loaded cells are able to eradicate tumoral cells in vitro and in vivo without significant toxicity. Also Ki67 expression, which is usually correlated with proliferation, is reduced after treatment. This approach enhances the effectiveness of the treatment when compared to just the enhanced permeation and retention effect (EPR) of the HGNs by themselves

Selective delivery of photothermal nanoparticles to tumors using mesenchymal stem cells as Trojan horses

et al.The main challenge of cancer treatment is to avoid or minimize systemic side effects in off-target tissues. Mesenchymal stem cells (MSCs) can be used as therapeutical carriers because of their ability to migrate and incorporate into inflammation areas including tumors. Here, this homing ability is exploited by carrying therapeutic nanoparticles (Hollow Gold Nanoparticles (HGNs)) following a >Trojan-horse> strategy. Amongst the different nanoparticles to be employed, HGNs have the capacity to resonate in the near infrared region when irradiated by an appropriated laser (808 nm). By transforming this absorbed energy into heat, they are capable to produce locally induced hyperthermia. At this wavelength healthy tissues have a minimal light absorption being the effect restricted to the tissues containing HGNs. By placing HGNs inside MSCs, the recognition, excretion and immune response are minimized. We demonstrate that MSCs internalize HGNs and reach the tumors still containing HGNs. After laser treatment this loaded cells are able to eradicate tumoral cells in vitro and in vivo without significant toxicity. Also Ki67 expression, which is usually correlated with proliferation, is reduced after treatment. This approach enhances the effectiveness of the treatment when compared to just the enhanced permeation and retention effect (EPR) of the HGNs by themselves.Financial support from the EU thanks to the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM) is gratefully acknowledged.Peer Reviewe

Upconverting carbon nanodots from ethylenediaminetetraacetic acid (EDTA) as near‐infrared activated phototheranostic agents

This work describes the synthesis of nitrogen‐doped carbon nanodots (CNDs) synthesized from ethylenediaminetetraacetic acid (EDTA) as a precursor and their application as luminescent agents with a dual‐mode theranostic role as near‐infrared (NIR) triggered imaging and photodynamic therapy agents. Interestingly, these fluorescent CNDs are more rapidly and selectively internalized by tumor cells and exhibit very limited cytotoxicity until remotely activated with a NIR illumination source. These CNDs are excellent candidates for phototheranostic purposes, for example, simultaneous imaging and therapy can be carried out on cancer cells by using their luminescent properties and the in situ generation of reactive oxidative species (ROS) upon excitation in the NIR range. In the presence of CNDs, NIR remote activation induces the in vitro killing of U251MG cells. Through the use of flow imaging cytometry, we have been able to successfully map and quantify the different types of cell deaths induced by the presence of intracellular superoxide anions (.O2−) and hydrogen peroxide (H2O2) ROS generated in situ upon NIR irradiation.The authors acknowledge the European Research Council for funding through an advanced grant research project (HECTOR grant number 267626; CADENCE grant number 742684) and a CIG‐Marie Curie Reintegration Grant (NANOLIGHT REA grant number 294094). M.C.O. acknowledges the Spanish Government for a FPU predoctoral fellowship.Peer reviewe

Integrated analysis of circulating immune cellular and soluble mediators reveals specific COVID19 signatures at hospital admission with utility for prediction of clinical outcomes

Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis. Methods: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity. Results: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFN alpha and TNF alpha, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNF alpha, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity. Conclusions: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death

Determination of the concentration of IgG against the spike receptor-binding domain that predicts the viral neutralizing activity of convalescent plasma and serum against SARS-CoV-2

4 figures, 1 table.Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Nevertheless, its efficacy in patients with COVID-19 remains uncertain. Thus, the establishment and validation of standardized methods that predict the viral neutralizing (VN) activity of plasma against SARS-CoV-2 is of utmost importance to appraise its therapeutic value. Using an in-house quantitative ELISA test and two independent cohorts with a total of 345 donors, we found that plasma and serum from most convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, with varying concentrations which correlate with previous disease severity and gender. Anti-RBD IgG plasma concentration significantly correlated with the plasma/serum VN activity against SARS-CoV-2 in vitro.Several hundred millions of people have been diagnosed of coronavirus disease 2019 (COVID-19), causing millions of deaths and a high socioeconomic burden. SARS-CoV-2, the causative agent of COVID-19, induces both specific T- and B-cell responses, being antibodies against the virus detected a few days after infection. Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Using an in-house quantitative ELISA test, we found that plasma from 177 convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, although at very different concentrations which correlated with previous disease severity and gender. Anti-RBD IgG plasma concentrations significantly correlated with the plasma viral neutralizing activity (VN) against SARS-CoV-2 in vitro. Similar results were found using an independent cohort of serum from 168 convalescent health workers. These results validate an in-house RBD IgG ELISA test in a large cohort of COVID-19 convalescent patients and indicate that plasma from all convalescent donors does not contain a high enough amount of anti-SARS-CoV-2-RBD neutralizing IgG to prevent SARS-CoV-2 infection in vitro. The use of quantitative anti-RBD IgG detection systems might help to predict the efficacy of the passive immunization using plasma from patients recovered from SARS-CoV-2.This research was funded by grants from the Aragon Government (Fondo COVID-19), the Fundacion Santander-Universidad de Zaragoza (Programa COVID-19), and the Instituto de Salud Carlos III (COV20-00308). Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón (Group B29_17R), Ministerio de Ciencia, Innovación y Universidades (MCNU), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R), Fundacion Inocente Inocente, ASPANOA, and Carrera de la Mujer de Monzón. Work in the RH-G lab is funded by ARAID, MCNU (CTQ2013-44367-C2-2-P, BFU2016-75633-P, and PID2019-105451GB-I00), and Gobierno de Aragón (E34_R17 and LMP58_18) with FEDER (2014–2020) funds for “Building Europe from Aragón. LS was supported by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM, SH and SR were supported by a PhD fellowship from Aragon Government. MA was supported by a post-doctoral fellowship “Juan de la Cierva-formación” from the Ministry of Science, Innovation and Universities and DdM by a post-doctoral fellowship “Sara Borrell”. JP was supported by the ARAID Foundation.Peer reviewe