Iron oxide nanocubes for magnetic hyperthermia

Durant la meva tesi s’han desenvolupat tres projectes principals, centrats en l'estudi de nanosistemes basats en l'ús de nanocubs d'òxid de ferro (IONCs) per a la hipertèrmia magnètica (HM). S’ha demostrat l'ús innovador de la configuració de HM per a l'oxidació suau de nanocubs tipus core-shell Fe1-xO/Fe3-δO4 a una única fase de Fe3O4. Atès que els valors de la taxa d'absorció específica (SAR) dels nanocubs tipus core-shell wüstita/magnetita han estat menors en comparació amb els IONCs de magnetita amb una longitud d’aresta de cub similar, el nucli de FeO s’ha oxidat mitjançant estimulació MH. Després de diversos tractaments amb HM, els valors de SAR s’han duplicat, mentre que l'estabilitat col·loïdal, la distribució de mida i la forma no s’han vist afectades. Els IONCs estimulats magnèticament han mostrat un valor de magnetització de saturació més elevat, reflectint canvis estructurals i de composició, confirmats a través d'estudis de microscòpia electrònica i de dispositius superconductors d'interferència quàntica. El tractament suau amb HM també ha fet possible l'ancoratge de molècules biològicament rellevants a la superfície dels nanocubs, preservant la seva activitat i millorant alhora el rendiment tèrmic dels IONCs. Els valors de SAR de IONCs tipus core-shell també s'han millorat acoblant els nanocubs en estructures de tipus cadena. Inicialment, es va desenvolupar l'agregació controlada dels IONCs durant la seva transferència en aigua, permetent la formació d'agregats (clústers) amb formats hidrodinàmics mitjans entre 30 i 100 nm. Així mateix, s’ha avaluat la resposta d’hipertèrmia de nanocubs individuals envers de nanoclústers col·loïdals tous de diferents mides. Les estructures denominades "dímers" i "trímers"—2D formades amb dos i tres IONCs—han mostrat valors SAR més alts. S’ha aconseguit la càrrega de fàrmac en dos nanosistemes dissenyats per alliberament de fàrmac quimioterapèutic desencadenat mitjançant calor. Tots dos sistemes s’han basat en IONCs de magnetita revestits amb polímers termo-responsius carregats amb doxorubicina. L'objectiu era obtenir nanotransportadors estables a la temperatura corporal que alliberessin la càrrega exclusivament sota l'aplicació d'un camp magnètic altern (AMF). D'aquesta manera, un cop obtinguts els IONCs individuals revestits amb polímer termo-responsiu amb altsTres proyectos principales se desarrollaron durante mi tesis centrada en el estudio de nanosistemas basados en el uso de nanocubos de óxido de hierro (IONCs) para la hipertermia magnética (HM). Se demostró el uso novedoso de la configuración de HM para la oxidación suave de nanocubos tipo core-shell Fe1-xO/Fe3-δO4 a una única fase de Fe3O4. Dado que los valores de la tasa de absorción específica (SAR) de los nanocubos tipo core-shell wüstita/magnetita fueron menores en comparación con los IONCs de magnetita con una longitud de borde de cubo similar, el núcleo de FeO se oxidó mediante estimulación MH. Después de varios tratamientos con HM, los valores de SAR se duplicaron, mientras que la estabilidad coloidal, la distribución del tamaño y la forma no se vieron afectadas. Los IONCs estimulados magnéticamente mostraron un valor de magnetización de saturación más elevado, reflejando cambios estructurales y de composición, confirmados a través de estudios de microscopía electrónica y de dispositivos superconductores de interferencia cuántica. El tratamiento suave con HM también hizo posible el anclaje moléculas biológicamente relevantes a la superficie de los nanocubos preservando su actividad y mejorando al mismo tiempo el rendimiento térmico de los IONCs. Los valores de SAR de IONCs tipo core-shell también se han mejorado ensamblando los nanocubos en estructuras de tipo cadena. Inicialmente, se desarrolló la agregación controlada de los IONCs durante su transferencia en agua, permitiendo la formación de agregados (clusters) con tamaños hidrodinámicos medios entre 30 y 100 nm. Asimismo, se evaluó la respuesta de hipertermia de nanocubos individuales frente a nanoclusters coloidales blandos de diferentes tamaños. Las estructuras denominadas "dímeros" y "trímeros"—2D formadas con dos y tres IONCs—mostraron valores SAR más altos. Se logró la carga de fármaco en dos nanosistemas diseñados para liberación de fármaco quimioterapéutico desencadenada mediante calor. Ambos sistemas se basaron en IONCs de magnetita revestidas con polímeros termo-responsivos cargados con doxorrubicina. El objetivo era obtener nanotransportadores estables a la temperatura corporal que liberaran la carga exclusivamente bajo la aplicación de un campo magnético alterno (AMF). De este modo, una vez obtenidos los IONCs individuales revestidos conThree main projects were conducted during my thesis that was focused on the study of nanosystems based on iron oxide nanocubes (IONCs) for magnetic hyperthermia (MH). The novel use of MH set-up for the mild oxidization of Fe1-xO/Fe3-δO4 core-shell nanocubes to single Fe3O4 phase was demonstrated. As specific absorption rate (SAR) values of wüstite/magnetite core-shell nanocubes were lower compared to magnetite IONCs of similar cube edge length, the FeO core was oxidized by MH stimulation. After several MH treatments, the SAR values increased twice, while colloidal stability, size distribution and shape remained unaffected. The magnetically stimulated IONCs showed higher saturation magnetization, reflecting structural and compositional changes, as confirmed by electron microscopy and superconductive quantum interference device studies. The mild MH treatment also opened up the possibility of attaching biologically relevant molecules to the surface of nanocubes and preserving their activity while improving the IONCs heat performance. The SAR values of core-shell IONCs were also enhanced by clustering the nanocubes in chain like structures. Initially, the controlled clustering of the IONCs during their water transfer was developed, enabling the formation of clusters with mean hydrodynamic sizes between 30 and 100 nm. The hyperthermia response of individual nanocubes vs. soft colloidal nanoclusters of different sizes was evaluated. The so called “dimers” and “trimers”—2D structures formed with two and three IONCs—showed higher SAR values. Drug loading on two nanosystems designed for heat-triggered chemotherapeutic drug release was achieved. Both systems were based on magnetite IONCs coated with thermo-responsive polymers loaded with doxorubicin. The goal was to have stable nanocarriers at body temperature that would release the cargo exclusively upon the application of an alternating magnetic field (AMF). Once individually thermo-responsive polymer coated IONCs with high SAR values were obtained, the heat-triggered doxorubicin release under AMF—at biologically relevant field conditions—was qualitatively, but not quantitatively proven

Mass spectrometry-based fragmentation as an identification tool in lignomics

The ensemble of all phenolics for which the biosynthesis is coregulated with lignin biosynthesis, i.e., metabolites from the general phenylpropanoid, monolignol, and (neo)-lignan biosynthetic pathways and their derivatives, as well as the lignin oligomers, is coined the lignome. In lignifying tissues, the lignome comprises a significant portion of the metabolome. However, as is true for metabolomics in general, the structural elucidation of unknowns represents the biggest challenge in characterizing the lignome. To minimize the necessity to purify unknowns for NMR analysis, it would be desirable to be able to extract structural information from liquid chromatography-mass spectrometry data directly. However, mass spectral libraries for metabolomics are scarce, and no libraries exist for the lignome. Therefore, elucidating the gas-phase fragmentation behavior of the major bonding types encountered in lignome-associated molecules would considerably advance the systematic characterization of the lignome. By comparative MS" analysis of a series of molecules belonging to the beta-aryl ether, benzodioxane, phenylcoumaran, and resinol groups, we succeeded in annotating typical fragmentations for each of these bonding structures as well as fragmentations that enabled the identification of the aromatic units involved in each bonding structure. Consequently, this work lays the foundation for a detailed characterization of the lignome in different plant species, mutants, and transgenics and for the MS-based sequencing of lignin oligomers and (neo)lignans

Asymmetric Assembling of Iron Oxide Nanocubes for Improving Magnetic Hyperthermia Performance

Magnetic hyperthermia (MH) based on magnetic nanoparticles (MNPs) is a promising adjuvant therapy for cancer treatment. Particle clustering leading to complex magnetic interactions affects the heat generated by MNPs during MH. The heat efficiencies, theoretically predicted, are still poorly understood because of a lack of control of the fabrication of such clusters with defined geometries and thus their functionality. This study aims to correlate the heating efficiency under MH of individually coated iron oxide nanocubes (IONCs) versus soft colloidal nanoclusters made of small groupings of nanocubes arranged in different geometries. The controlled clustering of alkyl-stabilized IONCs is achieved here during the water transfer procedure by tuning the fraction of the amphiphilic copolymer, poly(styrene-co-maleic anhydride) cumene-terminated, to the nanoparticle surface. It is found that increasing the polymer-to-nanoparticle surface ratio leads to the formation of increasingly large nanoclusters with defined geometries. When compared to the individual nanocubes, we show here that controlled grouping of nanoparticles - so-called "dimers" and "trimers" composed of two and three nanocubes, respectively - increases specific absorption rate (SAR) values, while conversely, forming centrosymmetric clusters having more than four nanocubes leads to lower SAR values. Magnetization measurements and Monte Carlo-based simulations support the observed SAR trend and reveal the importance of the dipolar interaction effect and its dependence on the details of the particle arrangements within the different clusters

Maize specialized metabolome networks reveal organ-preferential mixed glycosides

Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural characterization of 427 of the 5,420 profiled compounds, including phenylpropanoids, flavonoids, benzoxazinoids, and auxin-related compounds, among others. Only 75 of the 427 compounds were already described in maize. Analysis of the CSPP networks showed that phenylpropanoids are present in all organs, whereas other metabolic classes are rather organ-enriched. Frequently occurring CSPP mass differences often corresponded with glycosyl- and acyltransferase reactions. The interplay of glycosylations and acylations yields a wide variety of mixed glycosides, bearing substructures corresponding to the different biochemical classes. For example, in the tassel, many phenylpropanoid and flavonoid-bearing glycosides also contain auxin-derived moieties. The characterized compounds and mass differences are an important step forward in metabolic pathway discovery and systems biology research. The spectral metadata of the 5,420 compounds is publicly available (DynLib spectral database, https://bioit3.irc.ugent.be/dynlib/)

Degradation of lignin β-aryl ether units in Arabidopsis thaliana expressing LigD, LigF and LigG from Sphingomonas paucimobilis SYK-6

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the beta-aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized beta-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided

Uncovering the magnetic particle imaging and magnetic resonance imaging features of iron oxide nanocube clusters

Multifunctional imaging nanoprobes continue to garner strong interest for their great potential in the detection and monitoring of cancer. In this study, we investigate a series of spatially arranged iron oxide nanocube-based clusters (i.e., chain-like dimer/trimer, centrosymmetric clusters, and enzymatically cleavable two-dimensional clusters) as magnetic particle imaging and magnetic resonance imaging probes. Our findings demonstrate that the short nanocube chain assemblies exhibit remarkable magnetic particle imaging signal enhancement with respect to the individually dispersed or the centrosymmetric cluster analogues. This result can be attributed to the beneficial uniaxial magnetic dipolar coupling occurring in the chain-like nanocube assembly. Moreover, we could effectively synthesize enzymatically cleavable two-dimensional nanocube clusters, which upon exposure to a lytic enzyme, exhibit a progressive increase in magnetic particle imaging signal at well-defined incubation time points. The increase in magnetic particle imaging signal can be used to trace the disassembly of the large planar clusters into smaller nanocube chains by enzymatic polymer degradation. These studies demonstrate that chain-like assemblies of iron oxide nanocubes offer the best spatial arrangement to improve magnetic particle imaging signals. In addition, the nanocube clusters synthesized in this study also show remarkable transverse magnetic resonance imaging relaxation signals. These nanoprobes, previously showcased for their outstanding heat performance in magnetic hyperthermia applications, have great potential as dual imaging probes and could be employed to improve the tumor thermo-therapeutic efficacy, while offering a readable magnetic signal for image mapping of material disassemblies at tumor sites

Genome-Wide Transcriptomic and Proteomic Analyses of Bollworm-Infested Developing Cotton Bolls Revealed the Genes and Pathways Involved in the Insect Pest Defence Mechanism

Cotton bollworm, Helicoverpa armigera, is a major insect pest that feeds on cotton bolls causing extensive damage leading to crop and productivity loss. In spite of such a major impact, cotton plant response to bollworm infection is yet to be witnessed. In this context, we have studied the genome-wide response of cotton bolls infested with bollworm using transcriptomic and proteomic approaches. Further, we have validated this data using semi-quantitative real-time PCR. Comparative analyses have revealed that 39% of the transcriptome and 35% of the proteome were differentially regulated during bollworm infestation. Around 36% of significantly regulated transcripts and 45% of differentially expressed proteins were found to be involved in signalling followed by redox regulation. Further analysis showed that defence-related stress hormones and their lipid precursors, transcription factors, signalling molecules, etc. were stimulated, whereas the growth-related counterparts were suppressed during bollworm infestation. Around 26% of the significantly up-regulated proteins were defence molecules, while \u3e 50% of the significantly down-regulated were related to photosynthesis and growth. Interestingly, the biosynthesis genes for synergistically regulated jasmonate, ethylene and suppressors of the antagonistic factor salicylate were found to be up-regulated, suggesting a choice among stress-responsive phytohormone regulation. Manual curation of the enzymes and TFs highlighted the components of retrograde signalling pathways. Our data suggest that a selective regulatory mechanism directs the reallocation of metabolic resources favouring defence over growth under bollworm infestation and these insights could be exploited to develop bollworm-resistant cotton varieties

Spherical and needle shaped magnetic nanoparticles for friction and magnetic stimulated transformation of microorganisms

Supplementary material related to this article can be found online at https://doi.org/10.1016/j.nanoso.2021.100732.Spherical and needle shaped magnetic nanoparticles (MNPs) were synthesized by thermal decomposition, functionalized with 2-pyrrolidinone for the attachment of pUC19 plasmidic DNA and used in transformations assays of Escherichia coli JM109. Frictional and magnetic stimulation were employed for promoting the translocation of the nanoparticle-pUC19 complexes across the cell envelope. Transformants were obtained through frictional stimulation using needle shaped MNPs achieving a maximum transformation efficiency of 3.1 × 102 CFU/g pUC19. Magnetic stimulation was also performed using both types of nanoparticles under conventional magnetofection conditions on a magnetic bioreactor and did not induce transformation of E. coli JM109, possibly due to the field intensity at the region of the cells (100 mT) not being high enough to overcome the rigidity of the bacterial cell envelope. This work substantiates the need for the delivery agent to have a high aspect ratio in order to achieve transformation of prokaryotes. Moreover, it highlights the limitations of magnetic stimulation for translocation of MNPs across the microbial cell wall, as opposed to magnetofection of eukaryotic cells whereby the entry of genetic material can be readily accomplished using spherical MNPs through an endocytotic uptake mechanism.This work was supported by the Portuguese Foundation for Science and Technology (FCT) and the EU fund FEDER (Pro gram COMPETE) under projects PTDC/AMB/68393/2006, PEstOE/EQB/LA0023/2013, UID/FIS/04650/2021, RECI/BBB-EBI/0179/2012 and the Project ‘‘BioEnv - Biotechnology and Bioengineering for a sustainable world’’. The authors also acknowledge FCT for the fellowship SFRH/BD/71661/2010 awarded to Gabriel Mendes under the scope of the MIT-Portugal Program, nanoTherics for providing the Magnefect-Nano IITM device and Paul Brown for help in obtaining ATR-FTIR spectra. Finally, the authors acknowl edge funding by the Spanish State Research Agency (AEI) through the project PID2019-106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry and Education Depart ments under the ELKARTEK and PIBA (PIBA-2018-06) programs, respectively. All authors have given approval to the final version of the manuscript.info:eu-repo/semantics/publishedVersio