In situ nanofabrication of hybrid PEG-dendritic–inorganic nanoparticles and preliminary evaluation of their biocompatibility

An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybridsC.S.E. and A.G.-F. thank to the European Commission BIOCAPS (316265, FP7/REGPOT-2012-2013.1) and Xunta de Galicia (Agrupamento INBIOMED and Grupo con potencial crecimiento). A.S.-H. and E.F.-M. thank the Spanish Government (CTQ2012-34790) and the Xunta de Galicia (CN2011/037)S

Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250–450 nm and a positive zeta potential (~30–40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriersThis work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2011-30337-C02-02 and BIO2014-53091-C3-1-R). Financial support from the Xunta de Galicia (Centro singular de investigación de Galicia 2016–2019 and Grupo de referencia competitiva, ED431C 2016041) and the European Union (European Regional Development Fund—ERDF) is gratefully acknowledged. MP acknowledges fellowships from the Spanish Ministry of Education (FPU predoctoral grants program)S

A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier's surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC−), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAgThis work was supported by a grant from the Bill and Melinda Gates Foundation (www.gatesfoundation.org), Consolider Ingenio 2010 CSD2006-00012 (Ministry of Science and Innovation, Spain) and Competitive Reference Groups SUDOE-FEDER (SOE1/P1/E014). SV and MP acknowledge a fellowship from the Spanish Ministry of Education (FPU predoctoral grants). DWP was in part supported by Montana Agricultural Experiment Station and US Department of Agriculture Formula FundsS

Immunization with nanovaccines containing mutated K-Ras peptides and imiquimod aggravates heterotopic pancreatic cancer induced in mice

PurposeThe growing incidence and lethality of pancreatic cancer urges the development of new therapeutic approaches. Anti-tumoral vaccines can potentiate the immune response against the tumor, targeting specific antigens expressed only on tumor cells. In this work, we designed new vaccines for pancreatic cancer, composed by chitosan nanocapsules (CS NCs) containing imiquimod (IMQ) as adjuvant, and targeting the K-Ras mutation G12V.Experimental designWe tested the immunogenicity of our vaccines in mice, carrying different combinations of K-Ras mutated peptides. Then, we analyzed their prophylactic and therapeutic efficacy in mice bearing heterotopic pancreatic cancer.ResultsUnexpectedly, although good results were observed at short time points, the different combinations of our CS NCs vaccines seemed to potentiate tumor growth and reduce survival rate. We propose that this effect could be due to an inadequate immune response, partially because of the induction of a regulatory tolerogenic response.ConclusionOur results call for caution in the use of some NCs containing IMQ in the immunotherapy against pancreatic cancer

Pseudo-nitzschia Blooms in a Coastal Upwelling System: Remote Sensing Detection, Toxicity and Environmental Variables

The NW coast of the Iberian Peninsula is dominated by extensive shellfish farming, which places this region as a world leader in mussel production. Harmful algal blooms in the area frequent lead to lengthy harvesting closures threatening food security. This study developed a framework for the detection of Pseudo-nitzschia blooms in the Galician rias from satellite data (MERIS full-resolution images) and identified key variables that affect their abundance and toxicity. Two events of toxin-containing Pseudo-nitzschia were detected (up to 2.5 μg L−1 pDA) in the area. This study suggests that even moderate densities of Pseudo-nitzschia in this area might indicate high toxin content. Empirical models for particulate domoic acid (pDA) were developed based on MERIS FR data. The resulting remote-sensing model, including MERIS bands centered around 510, 560, and 620 nm explain 73% of the pDA variance (R2 = 0.73, p < 0.001). The results show that higher salinity values and lower Si(OH)4/N ratios favour higher Pseudo-nitzschia spp. abundances. High pDA values seem to be associated with relatively high PO43, low NO3− concentrations, and low Si(OH)4/N. While MERIS FR data and regionally specific algorithms can be useful for detecting Pseudo-nitzschia blooms, nutrient relationships are crucial for predicting the toxicity of these blooms

Cytotoxicity effects of metal oxide nanoparticles in human tumor cell lines

Metallic and metal oxide nanoparticles (Nps) have a wide range of applications in various settings including household, cosmetics and chemical industries, as well as for coatings. Nevertheless, an in-depth study of the potential toxic effects of these Nps is still needed, in order to fulfill the mandatory requirement of ensuring the safety of workers, patients and the general public. In this study, Quick Cell colorimetric assays were used to evaluate the in vitro toxicity of different metal oxide Nps [Fe(II,III)Ox, TiOx, ZnO and CeO2] in several cell lines. The ZnO Nps were found to be highly toxic, with a lethal dose ≥100 μg/ml for all the cell lines studied. Western blot was also used to test the ability of the different Nps to activate the complement pathway. However, no activation of this cascade was observed when the Nps were added. In addition, the aggregation state and charge of the Nps in culture media was studied by dynamic light scattering (DLS) and measurement of zeta potential. Transmission Electron Microscopy was used to analyze Np uptake and localization at the cellular level

A Comprehensive Study of Vesicular and Non-Vesicular miRNAs from a Volume of Cerebrospinal Fluid Compatible with Clinical Practice

Cerebrospinal fluid (CSF) microRNAs (miRNAs) have emerged as potential biomarkers for minimally invasive diagnosis of central nervous system malignancies. However, despite significant advances in recent years, this field still suffers from poor data reproducibility. This is especially true in cases of infants, considered a new subject group. Implementing efficient methods to study miRNAs from clinically realistic CSF volumes is necessary for the identification of new biomarkers. Methods: We compared six protocols for characterizing miRNAs, using 200-mu L CSF from infants (aged 0-7). Four of the methods employed extracellular vesicle (EV) enrichment step and the other two obtained the miRNAs directly from cleared CSF. The efficiency of each method was assessed using real-time PCR and small RNA sequencing. We also determined the distribution of miRNAs among different CSF shuttles, using size-exclusion chromatography. Results: We identified 281 CSF miRNAs from infants. We demonstrated that the miRNAs could be efficiently detected using only 200 mu L of biofluid in case of at least two of the six methods. In the exosomal fraction, we found 12 miRNAs that might be involved in neurodevelopment. Conclusion: The Norgen and Invitrogen protocols appear suitable for the analysis of a large number of miRNAs using small CSF samples.This work was supported by the Basque Government [IT989-16], the Spanish Ministry of Economy and Competitiveness MINECO [SAF2015066312], and the Ramon Areces Foundation [FRA-17-JMF]. We thank MINECO for the REDIEX (Spanish Excellence Network in Exosomes) and the Severo Ochoa Excellence Accreditation (SEV-2016-0644). Funding for open access charge: Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Lactoferrin-based nanoparticles as a vehicle for iron in food applications: development and release profile

This study aims at developing and characterizing bovine lactoferrin (bLf) nanoparticles as an iron carrier. bLf nanoparticles were characterized in terms of size, polydispersity index (PdI), electric charge (-potential), morphology, structure and stability over time. Subsequently, iron release experiments were performed at different pH values (2.0 and 7.0) at 37 °C, in order to understand the release mechanism. bLf (0.2%, w/v) nanoparticles were successfully produced by thermal gelation (75 °C for 20 min). bLf nanoparticles with 35 mM FeCl3 showed an iron binding efficiency value of approximately 20%. The nanoparticles were stable (i.e. no significant variation of size and PdI of the nanoparticles) for 76 days at 4 °C and showed to be stable between 4 and 60 °C and pH 2 and 11. Release experiments at pH 2 showed that iron release could be described by the linear superposition model (explained by Fick and relaxation phenomenon). On the contrary, the release mechanism at pH 7 cannot be described by either Fick or polymer relaxation behaviour. In general, results suggested that bLf nanoparticles could be used as an iron delivery system for future food applications.Joana T. Martins, Ana I. Bourbon and Ana C. Pinheiro acknowledge the Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/89992/2012, SFRH/BD/73178/2010 and SFRH/BPD/101181/2014). This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). This study was also supported by FCT under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01- 0124-FEDER-027462). The authors would like to acknowledge Cristina Quintelas and Filomena Costa from CEB, University of Minho for helping with AAS and ICP analysis, respectively. Also, the authors would like to thank Rui Fernandes from IBMC, University of Porto for assistance with TEM analysi

PAMAM dendrimers functionalised with an anti-TNF α antibody and chondroitin sulphate for treatment of rheumatoid arthritis

"Available online 6 January 2021"Rheumatoid arthritis is a chronic autoimmune disease characterised by joint synovial inflammation, along with cartilage and bone tissue destruction. Dendrimers can offer new opportunities as drug delivery systems of molecules of interest. Herein we aimed to develop poly(amidoamine) dendrimers (PAMAM), functionalised with chondroitin sulphate (CS), lined with anti-TNF α antibodies (Abs) to provide anti-inflammatory properties. Physicochemical characterisation demonstrated that anti-TNFα Abs-CS/PAMAM dendrimer NPs were successfully produced. The in vitro studies revealed that CS/PAMAM dendrimer NPs did not affect the ATDC5 and THP-1 cell lines' metabolic activity and proliferation, presenting good cytocompatibility and hemocompatibility. Moreover, anti-TNFα Abs-CS/PAMAM dendrimer NPs showed suitable TNF α capture capacity, making them appealing for new immunotherapies in RA patients.The authors thank the financial support under the Norte2020 project (“NORTE-08-5369-FSE-000044”) and BD/137726/2018/J6 21340zkMF. The FCT distinction attributed to J. M. O. under the Investigator FCT program (number IF/01285/2015) is also greatly acknowledged. C. G. also wished to acknowledge FCT for supporting her research (No. SFRH/BPD/94277/2013). RS and AG-F thank Xunta de Galicia (Grupo de Referencia Competitiva, ED431C 2016041) and Centro de Investigaciones Biom ́edicas (CINBIO), Vigo, Spain, for sup-porting their research

Lactoferrin-based nanohydrogel as a vehicle for iron delivery – preparation and release profile

Lactoferrin is a milk protein involved in numerous biological functions, such as regulation and transport of free iron levels, antioxidant and antimicrobial activities. Nanosystems, such as nanohydrogels, may be a vehicle to enrich foods with essential nutrients. Once iron deficit is a major nutritional problem, its inclusion in lactoferrin nanohydrogels could be one of the approaches to improve their stability, protection and subsequent absorption. Moreover, understanding the release mechanisms involved during human consumption, recurring to mathematical modeling, is important for the design of nanohydrogels carriers allowing foreseeing if the developed systems are appropriated to food products. The research conducted aims at developing bovine lactoferrin-based nanohydrogel, and understanding the transport mechanisms of the nanosystem used as iron vehicle. Nanohydrogel were formed by thermal gelation of lactoferrin (0.2 % (w/v)) at 75 °C for 20 minutes. After heat treatment, 35 mM (w/v) of ferric chloride was added. Nanohydrogels were characterized in terms of size distribution, polydispersity index (PDI) and morphology. Release experiments were conducted at different pH (2.0 or 7.0) at 37 o C (simulation of human digestive system conditions). Mathematical models were used to discuss the transport mechanism. Results showed that nanohydrogels with size of 110.0±0.4 nm and PDI of 0.218±0.005 were produced, and were stable during 76 weeks. Data from release experiments at pH 2 were successfully described by linear superimposition model which accounts for both Fick and Case II (polymer relaxation phenomenon) transport; in contrast, transport mechanism at pH 7 cannot be described by either Fick or Case II transport. These results suggest that lactoferrin nanohydrogel system can be used in the food industry as a carrier to facilitate the release of essential nutrients, such as iron, in the human body. Additionally, the approach presented in this work allows interpretation of the phenomena involved in mass transport at the nano-scale