Photoantimicrobial Biohybrids by Supramolecular Immobilization of Cationic Phthalocyanines onto Cellulose Nanocrystals

This is the peer-reviewed version of the following article: Anaya‐Plaza, E., van de Winckel, E., Mikkilä, J., Malho, J. M., Ikkala, O., Gulías, O., ... & Kostiainen, M. A. (2017). Photoantimicrobial biohybrids by supramolecular immobilization of cationic phthalocyanines onto cellulose nanocrystals. Chemistry–A European Journal, 23(18), 4320-4326., which has been published in final form at https://doi.org/10.1002/chem.201605285. This article may be used for non-commercial purposes in accordance with Wiley-VCH Terms and Conditions for Self-ArchivingThe development of photoactive and biocompatible nanostructures is a highly desirable goal to address the current threat of antibiotic resistance. Here, we describe a novel supramolecular biohybrid nanostructure based on the non-covalent immobilization of cationic zinc phthalocyanine (ZnPc) derivatives onto unmodified cellulose nanocrystals (CNC), following an easy and straightforward protocol, in which binding is driven by electrostatic interactions. These non-covalent biohybrids show strong photodynamic activity against S. aureus and E. coli, representative examples of Gram-positive and Gram-negative bacteria, respectively, and C. albicans, a representative opportunistic fungal pathogen, outperforming the free ZnPc counterparts and related nanosystems in which the photosensitizer is covalently linked to the CNC surfaceA.d.l.E. acknowledges a Ramón y Cajal contract from the Spanish Ministry of Economy (MINECO). The work at Madrid was supported by the EU [SO2S (FP7‐PEOPLE‐2012‐ITN, 316975); and CosmoPHOS‐nano (FP7‐NMP‐2012‐6, 310337‐2)], the Spanish MINECO [CTQ‐2014‐52869‐P (T.T.) and CTQ‐2014‐53673‐P (A.d.l.E.)] and Comunidad de Madrid [FOTOCARBON (S2013/MIT‐2841)]. J.M., V.L., and M.A.K. acknowledge support through the Emil Aaltonen Foundation and the Academy of Finland (grants 267497, 273645 and 263504). This work was supported by the Academy of Finland through its Centers of Excellence Programme (2014–2019) and made use of the Aalto University Nanomicroscopy Centre (Aalto NMC). The work in Barcelona was supported by the Spanish MINECO (grant CTQ2013‐48767‐C3‐1‐R). R.B.‐O. thanks the European Social Funds and the SUR del DEC de la Generalitat de Catalunya for his predoctoral fellowship (2016 FI B1 00021)

Bio-orgaanisten hybridirakenteiden tutkimuksia

The advances of supramolecular chemistry, bottom-up fabrication and bioinspired approaches have opened pathways to well-defined, hierarchical and functional materials at nanometer scale. Biological systems master the fabrication of near-perfect macromolecules and organized bulk materials from simple components under mild conditions. For self-assembly and material applications these biomaterials offer precision, diversity and functionality. This thesis demonstrates how biological and synthetic organic materials can be combined into bio-organic hybrid assemblies to promote biomedical and composite applications. In publication I multivalent, low-molecular-weight dendrons with cleavable disulfide bonds were synthesized and used for the electrostatic binding and triggered release of DNA. The dendrons bound DNA in a generation-dependent fashion and reduction of the disulfide bonds rapidly liberated the DNA. Degradable multivalent binding has been shown to enhance gene delivery, as the reducing cytosolic environment induces the release of DNA. In publication II the reducible dendrons were combined with photocleavable dendrons for the controlled formation of nanoscale DNA origamis. Binding of the origami constituents prevented their self-assembly into origamis, which was subsequently initiated by externally triggered degradation of the dendrons. Publication III studied the co-assembly of anionic icosahedral viruses and amphiphilic Janus dendrimers. Tuning of the dendrimer structures and the electrolyte concentration provided superlattices with a face-centered cubic unit cell. The assemblies resembled natural viral inclusion bodies, and provide means for studying their self-assembly processes. In publication IV rod-like cellulose nanocrystals (CNC) were grafted with a cationic polymer. The cationic CNCs efficiently assembled the anionic icosahedral viruses. Additionally, the CNCs enabled the concentration and extraction of the viruses from solution. The assemblies can be further transferred to biomedical applications and allow the recovery of viruses via electrolyte-gated release. In publication V the CNCs were functionalized with cross-linkable alkyl chains and used for the reinforcement of a synthetic rubber. The moisture-repellent composites showed a hierarchical structure, where the CNCs assembled into ordered biomimetic structures at high weight fractions. Simultaneously the stress-strain behavior abruptly changed from nonlinear rubbery to almost linear with increasing CNC fraction, while the tensile modulus increased by almost two orders of magnitude.Supramolekyylikemian, itsejärjestymisen ja luontoa imitoivien lähestymistapojen kehittyminen on mahdollistanut hyvin määriteltyjen, hierarkkisten ja funktionaalisten nanomateriaalien valmistuksen. Luonnossa lähes täydelliset makromolekyylit ja järjestyneet materiaalit muodostuvat yksinkertaisista rakenneosista miedoissa olosuhteissa. Materiaalitutkimukseen nämä biomateriaalit tuovat uutta täsmällisyyttä, monimuotoisuutta ja toiminnallisuutta. Väitöskirjassa osoitetaan, kuinka biologiset ja synteettiset orgaaniset materiaalit voidaan yhdistää hybridimateriaaleiksi hyödyttäen lääketiedettä ja komposiittitutkimusta. Julkaisussa I valmistettiin monivalentteja, pelkistämällä hajotettavia disulfidisidoksia sisältäviä dendroneita ja tutkittiin niiden kykyä sitoa DNA:ta ionisilla vuorovaikutuksilla ja vapauttaa se ulkoisen signaalin avulla. Dendronien affiniteetti DNA:ta kohtaan riippui niiden valenssista, ja pelkistys vapautti DNA:n nopeasti. Menetelmä voi tehostaa geeniterapiaa, jossa solujen pelkistävä ympäristö hajottaa DNA-dendroni –kompleksit. Julkaisussa II pelkistettäviä ja valon avulla hajotettavia dendroneita käytettiin DNA-origamien muodostumisen hallintaan. Origamien rakenneosien sitominen dendroneilla esti niiden itsejärjestymisen, joka voitiin käynnistää hajottamalla dendronit ulkoisilla signaaleilla. Lisäksi dendronit kokosivat ja vapauttivat hallitusti kokonaisia origameja. Julkaisu III tutki anionisten, ikosaedrin muotoisten virusten ja amfifiilisten Janus-dendrimeerien järjestymistä. Dendrimeerien rakenteiden ja elektrolyyttikonsentraatioiden säätely tuotti superhiloja pintakeskisellä kuutiollisella kiderakenteella. Luonnossa esiintyviä inkluusiokappaleita muistuttavat kiteet voivat edesauttaa niiden tutkimusta. Julkaisussa IV kationista polymeeriä oksastettiin sauvamaisten selluloosan nanokiteiden pinnalta. Geeninsiirtoa tehostavia peptidikuituja muistuttavat kationiset selluloosakiteet sitoivat anionisia viruksia tehokkaasti ja mahdollistivat niiden eristämisen liuoksesta. Komplekseista virukset voidaan siirtää jatkotutkimuksiin tai vapauttaa nostamalla elektrolyyttikonsentraatiota. Julkaisussa V selluloosan nanokiteisiin liitettiin hydrofobisia, ristisilloitettavia alkyyliketjuja, ja niillä vahvistettiin synteettistä kumia. Nanokiteet järjestyivät kosteutta hylkivissä komposiiteissa suurissa pitoisuuksissa biomimeettisiksi orientoituneiksi ja hierarkkisiksi kerrosrakenteiksi. Nanokiteiden määrän kasvaessa komposiittien jännitys-venymä –käyrä muuttui äkillisesti kumimaisesta lähes lineaariseksi, kimmokertoimen kasvaessa lähes kaksi kertaluokkaa

De la traducción a la innovación en la escuela 2.0 : un estudio de caso de los docentes del CEIP Manuel Siurot de Huelva

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Transition to Reinforced State by Percolating Domains of Intercalated Brush-Modified Cellulose Nanocrystals and Poly(butadiene) in Cross-Linked Composites Based on Thiol–ene Click Chemistry

The classic nanocomposite approach aims at percolation of low fraction of exfoliated individual reinforcing nanoscale elements within a polymeric matrix. By contrast, many of the mechanically excellent biological nanocomposites involve self-assembled and space-filled structures of hard reinforcing and soft toughening domains, with high weight fraction of reinforcements. Here we inspect a new concept toward mimicking such structures by studying whether percolation of intercalated domains consisting of alternating rigid and reinforcing, and soft rubbery domains could allow a transition to a reinforced state. Toward that, we present the functionalization of rigid native cellulose nanocrystals (CNCs) by esterification with a dense hydrocarbon chain brush containing cross-linkable double bonds. Composite films with 0–80 wt % of such modified CNCs (mCNCs) within a poly­(butadiene) (PBD) rubber matrix were prepared via cross-linking by UV-light initiated thiol–ene click reaction. Transmission electron microscopy showed structures at two length scales, where the mCNCs and PBD form domains having internal aligned self-assemblies of alternating hard mCNCs and soft PBD with periodicity of ca. 40 nm, and where additional PBD connects such domains. Increasing the weight fraction of mCNCs causes an uncommon abrupt transition from PBD-dominated soft materials to significantly reinforced mCNC-dominated mechanical properties, suggesting that the intercalated self-assembled mCNC/PBD domains percolate in PBD upon passing 30–35 wt % of mCNCs. Maximum stress of 16 MPa at mCNC fraction of 80 wt % was obtained. The mechanical properties of the composites show exceptional insensitivity to air humidity. The shown simple concept of percolative intercalated nanocomposites suggests searching for more general biomimetic compositions involving several deformation mechanisms for improved mechanical properties

Hierarchically Ordered Supramolecular Protein-Polymer Composites with Thermoresponsive Properties

Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc, Fm3¯¯¯m) Bravais lattice where lattice parameter a = 18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion.Peer reviewe

Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.acceptedVersionPeer reviewe

Generic Method for Modular Surface Modification of Cellulosic Materials in Aqueous Medium by Sequential “Click” Reaction and Adsorption

A generic approach for heterogeneous surface modification of cellulosic materials in aqueous medium, applicable for a wide range of functionalizations, is presented. In the first step, carboxymethyl cellulose (CMC) modified with azide or alkyne functionality, was adsorbed on a cellulosic substrate, thus, providing reactive sites for azide–alkyne cycloaddition click reactions. In the second step, functional units with complementary click units were reacted on the cellulose surface, coated by the click-modified CMC. Selected model functionalizations on diverse cellulosic substrates are shown to demonstrate the generality of the approach. The concept by sequentially combining the robust physical adsorption (“physical click”) and robust chemical reaction (“chemical click”) allows versatile, simple, and environmentally friendly modification of a cellulosic substrate with virtually any azide- or alkyne-modified molecule and even functionalization with several types of units