Síntese de nanopartículas magnéticas de Poli(ureia-uretano) e aplicação como suporte na imobilização da lipase B de Candida antarctica

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2015.Materiais superparamagnéticos encapsulados em matriz polimérica são interessantes em diversos tipos de aplicações, incluindo áreas biomédicas, processos de separação, biotecnologia e imobilização de enzimas. Neste trabalho, nanopartículas superparamagnéticas de poli(ureia-uretano), PUU, foram obtidas via polimerização interfacial em miniemulsão e utilizadas como suporte na imobilização da lipase B de Candida antarctica. Nanopartículas de magnetita com comportamento superparamagnético foram obtidas pelo método de co-precipitação e, após a síntese, as nanopartículas magnéticas, NPMs, foram recobertas com ácido oleico, AO, para a obtenção de uma superfície hidrofóbica. A síntese de nanopartículas de PUU foi conduzida utilizando 1,6-hexanodiol como poliol e diisocinato de isoforona, IPDI, como diisocianato. As NPMs-AO foram incorporadas em PUU durante a polimerização interfacial em miniemulsão. Nanopartículas de PUU com NPMs-AO incorporadas foram caracterizadas por Espectroscopia de infravermelho com transformada de Fourier, (FTIR), análise termogravimétrica, (TGA), magnetometria de amostra vibrante, (MAV), microscopia eletrônica de transmissão, (MET), e microscopia eletrônica de varredura, (MEV-FEG). Os resultados mostraram alta eficiência de encapsulação e as análises de MAV confirmaram que NPMs-AO apresentam comportamento superparamagnético após a encapsulação. Lipase B de Candida antarctica foi imobilizada nas nanopartículas poliméricas magnéticas de PUU, (NPMs PUU), em uma única etapa durante a polimerização interfacial em miniemulsão e o derivado atraído por um campo magnético externo foi utilizado como catalisador em reações de síntese de etil oleato, geranil oleato e geranil propionato. O teor de ácidos graxos livres nas amostras, medido pelo método de titulação, mostrou que conversões em ésteres acima de 85% foram obtidas para todos os sistemas estudados. A análise de microscopia óptica de fluorescência mostrou a presença de lipase na superfície das NPMs-PUU, confirmando a formação de ligação entre enzima-suporte. Em um segundo método de imobilização, lipase B de Candida antarctica foi imobilizada em NPMs PUU em duas etapas após a polimerização interfacial em miniemulsão, com as partículas liofilizadas. O processo foi realizado pelo contato da enzima com o suporte em tampão fosfato pH 7,6, a solução foi incubada em shaker a 30 °C a 150 rpm e a atividade de esterificação enzimática foi medida. Atividades relativas acima de 85% em relação à enzima livre foram obtidas em uma hora deimobilização com concentração de enzima de 0,55 mg/mL. Análises de FTIR, MEV-FEG, estabilidade térmica (40, 60 e 80 °C), estabilidade ao pH (4, 7 e 10) e estabilidade ao armazenamento mostraram a eficiência de imobilização da lipase por adsorção em NPMs PUU, que apresentou estabilidade em diferentes condições reacionais.Abstract : Superparamagnetic materials encapsulated in a polymeric matrix are interesting for several applications, including biomedical, separation process, biotechnology and enzyme immobilization. In this work, superparamagnetic poly(urea-urethane), PUU, nanoparticles were obtained by interfacial miniemulsion polymerization and used as a support for the immobilization of lipase B from Candida antarctica (CALB). Magnetite nanoparticles presenting superparamagnetic behavior were obtained by the co-precipitation method and after the synthesis the magnetic nanoparticles, MNPs, were coated with oleic acid, OA, to provide a hydrophobic surface. The synthesis of PUU nanoparticles was performed using 1,6-hexanediol as polyol and isophorone diisocyanate, IPDI, as diisocyanate. The MNPs-OA were incorporated during the interfacial miniemulsion polymerization. PUU with MNPs-OA were characterized by Fourier transform infrared spectroscopy, (FTIR), thermogravimetric analysis, (TGA), electromagnetic vibrating sample magnetometry, (VSM), transmission electron microscopy, (TEM), and scanning electron microscopy, (SEM-FEG). Results showed a high encapsulation efficiency and VSM analysis showed that PUU with MNPs-OA still presented superparamagnetic behavior after the encapsulation step. CALB was immobilized on magnetic poly(urea-urethane) nanoparticles, (MNPs PUU) in a single step during the interfacial miniemulsion polymerization. After the immobilization process, the immobilized enzyme was attracted by an external magnetic field and was used as a catalyst in the synthesis of ethyl oleate, geranyl propionate and geranyl oleate. Ester conversions above 85% were obtained for all systems based on the free fatty acids contents measured by titration. Fluorescence microscopy images confirmed the immobilization of enzyme onto MNPs-PUU. CALB was immobilized on MNPs PUU in two steps after the interfacial polymerization in miniemulsion. Lipase was added to lyophilized particles (MNPs PUU) in phosphate buffer (0.1 M, pH 7.6). The mixture was incubated at 30 °C in a shaker for 30-360 min to determine the time required for maximum immobilization efficiency and the enzyme activity was determined by the esterification reactions between lauric acid and propanol. The relative activities of the immobilized enzyme in relation to free enzyme were above 85% in one hour with 0.55 mg/mL of enzyme in solution. FTIR analysis, SEM-FEG, thermal stability (40, 60 and 80 °C), pH stability (4, 7 e 10) and storagestability showed the efficiency of the method, that allowed to maintain high stability of the immobilized enzyme under different conditions

Improving the Performance of Photoactive Terpene-Based Resin Formulations for Light-Based Additive Manufacturing

Photocurable liquid formulations have been a key research focus for the preparation of mechanically robust and thermally stable networks. However, the development of renewable resins to replace petroleum-based commodities presents a great challenge in the field. From this perspective, we disclose the design of photoactive resins based on terpenes and itaconic acid, both potentially naturally sourced, to prepare photosets with adjustable thermomechanical properties. Biobased perillyl itaconate (PerIt) was synthesized from renewable perillyl alcohol and itaconic anhydride via a scalable solvent-free method. Photoirradiation of PerIt in the presence of a multiarm thiol and photoinitiator led to the formation of networks over a range of compositions. Addition of nonmodified terpenes (perillyl alcohol, linalool, or limonene) as reactive diluents allowed for more facile preparation of photocured networks. Photosets within a wide range of properties were accessed, and these could be adjusted by varying diluent type and thiol stoichiometry. The resins showed rapid photocuring kinetics and the ability to form either brittle or elastic materials, with Young’s modulus and strain at break ranging from 3.6 to 358 MPa and 15 to 367%, respectively, depending on the chemical composition of the resin. Glass transition temperatures (Tg) were influenced by thioether content, with temperatures ranging from 5 to 43 °C, and all photosets displayed good thermal resistance with Td,5% > 190 °C. Selected formulations containing PerIt and limonene demonstrated suitability for additive manufacturing technologies and high-resolution objects were printed via digital light processing (DLP). Overall, this work presents a simple and straightforward route to prepare renewable resins for rapid prototyping applications

Polyesters with main and side chain phosphoesters as structural motives for biocompatible electrospun fibres

Phosphoester containing polymers are promising materials in biomedical applications due to their biocompatibility and biodegradability. Utilising thiol-ene chemistry, the synthesis of two novel structural polymer motives combining polyesters and phophoester groups was explored. The first polymer was obtained by coupling ene-functional poly(thioether-phosphoester) with thiol functional poly(pentadecalactone). While the coupling reaction was successful, yields remained low presumably due to inadequate endgroup stoichiometry. The second polymer comprised phosphoester side groups conjugated to unsaturated poly(globalide). Double bond conversions up to 84% were achieved depending of the type of phosphoester thiol and relative reactant ratios. The resulting polymers transitioned from solid semicrystaline to liquid amorphous with increasing degree of phosphoester conjugation. Electrospun fibres from polymers with 14% phosphoester conjugation allowed attachment and survival of human dermal fibroblasts, indicating their biocompatibility. These polymers represent a new class of easily accessible biocompatible polyester-phosphoester hybrid materials as potential building blocks for tunable biomaterials

Unsaturated macrolactone polymerization followed by its modification and crosslinking via click chemistry-based reactions

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2019.Poliésteres alifáticos estão entre os polímeros biodegradáveis mais utilizados em aplicações biomédicas. A síntese de poliésteres pode ser conduzida por duas rotas principais: polimerização por abertura de anel e policondensação. Neste trabalho, o poliéster poli(globalide) foi sintetizado via polimerização enzimática por abertura de anel em solução e em miniemulsão a partir da macrolactona insaturada globalide. O efeito da concentração de enzima na e-ROP em solução utilizando Novozym 435 (NVZ 435, lipase B de Candida antarctica imobilizada em suporte poroso) mostrou que as massas molares não foram alteradas (de forma acentuada) quando utilizadas diferentes concentrações de enzima, permanecendo em torno de 30.000 g mol-1 (Mw). As reações conduzidas em solução com a lipase NS88011 (lipase B de Candida antarctica imobilizada em suporte mais barato quando comparado com a lipase NVZ435) mostraram que maiores massas molares (Mw 60.500 g mol-1) podem ser obtidas quando esta enzima é utilizada na e-ROP, em comparação aos poliésteres obtidos com a Novozym 435, e períodos de reação mais longos são necessários para a obtenção de rendimentos na faixa de 80-90%. Lipases livres (CALB e NS40116) foram utilizadas para a obtenção de nanopartículas de PGl via polimerização enzimática por abertura de anel (e-ROP) em miniemulsão. Diâmetros na faixa de 70 a 280 nm foram obtidos quando CALB foi utilizada como biocatalisador e massas molares na faixa entre 6.800 a 23.200 g mol-1 foram atingidas. Entretanto, nanopartículas sintetizadas com NS40116 mostraram-se instáveis e apenas diâmetros acima do limite de detecção do equipamento foram encontrados (6 µm). Após a síntese e caracterização de PGl via e-ROP em solução e miniemulsão, duas estratégias diferentes de click-chemistry foram utilizadas para a modificação da cadeia polimérica. A primeira estratégia utilizada foi a modificação pós-polimerização de PGl via reações tiol-eno. Três tióis contendo grupamentos fosfoéster foram sintetizados em tolueno para obter compostos puros com rendimentos acima de 60% após purificação em coluna de silica. Assim, os tióis sintetizados foram adicionados na cadeia de PGl e parâmetros como razão molar tiol:eno e concentração de iniciador foram avaliados. Resultados de conversão em dupla ligação e propriedades térmicas dos polímeros foram afetados pelo tamanho da cadeia do tiol utilizado. Quando TF2 foi adicionado na cadeia de PGl, conversões em dupla ligação de 14 a 84% foram obtidas e o polímero com 14% de modificação foi escolhido para produzir fibras via eletrofiação. Fibras homogêneas e sem a presença de grânulos na faixa de 11 µm foram obtidas e este material mostrou-se não tóxico em células de fibroblastos. Em uma segunda metodologia, a cadeia polimérica de PGl foi modificada ou reticulada via reações com triazolinedionas (TADs). PGl foi modificado com TAD monofuncional (4-phenyl-1,2,4-triazoline-3,5-dione) para obter polímeros com elevado grau de modificação (97%) em reação conduzida em THF em um sistema livre de catalisador e iniciadores. Com o intuito de obter scaffolds porosos, fibras de PGl foram obtidas por eletrofiação e reticuladas com TADs bifuncionais para uma melhoria nas propriedades mecânicas do material. De uma forma geral, a química de triazolinedionas mostrou-se eficiente para introduzir funcionalidades e reticulações na cadeia de PGl e modificar as propriedades térmicas e mecânicas do polímero.Abstract: Aliphatic polyesters are among the most used biodegradable polymers in biomedical applications. Polyesters synthesis can be conducted by two main routes: polycondensation and ring-opening polymerization. In this work, poly(globalide) (PGl) polyester was synthesized by enzymatic ring-opening polymerization in solution and miniemulsion from the unsaturated macrolactone globalide (Gl). Polymerization kinetics of Gl e-ROP in solution showed that different enzyme concentrations did not affect polymer molecular weight when Novozym 435 (NVZ 435, Candida antarctica B lipase immobilized on a porous support) was used as biocatalyst and, molecular weights in a range of 30,000 g mol-1 (Mw) were obtained. On the contrary, when NS88011 (Candida antarctica B lipase immobilized on a cheaper support than NVZ 435) was used as biocatalyst, the molecular weights and yields were highly affected by the enzyme concentration and the kinetics were slower than in the NVZ435 case. However, higher molecular weights were reached when NS88011 was used. Free lipases (CALB and NS40116) were used to conduct miniemulsion e-ROP of Gl. PGl nanoparticles diameters from 70 to 280 nm and molecular weights ranging from 6,800 to 23,200 g mol-1 were obtained with CALB. Nanoparticles formed by the NS40116 catalyzed system were non-stable under the tested conditions and diameters were always above the measurement limit of the dynamic light scattering equipment (6 µm). After PGl synthesis and characterization via e-ROP in solution and miniemulsion, two different strategies were used to modify their structure via click chemistry. In the first approach, post-polymerization modification of PGl was conducted via thiol-ene reactions. Three different thiols containing phosphoester groups were synthesized in toluene resulting in yields greater than 60% after silica column purification. After that, these compounds were attached to the poly(globalide) backbone and the effects of thiol:ene ratio and initiator concentration were evaluated. Double bound conversions and polymer thermal properties were related to the thiol chain length. When diethyl 6-mercapto-1-hexyl phosphate was added to PGl chain, double bond conversions from 14 to 84% were reached. Modified polymer with 14% of double bond conversion was processed to obtain fibers with diameters around 11 µm via electrospinning, which were non-toxic to fibroblast cells. In the second approach, PGl chain was either modified or crosslinked via triazolinedione (TAD) reactions. PGl was modified with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to obtain high degree of modification (97%) in a reaction conducted with THF as solvent and without catalyst or initiator. In order to obtain porous scaffolds, plain PGl fibers were obtained by electrospinning and crosslinked with two different bifunctional TAD molecules (hexamethylene bis-TAD or MDP bis-TAD) to tune the mechanical properties of the fibers. In summary, TAD chemistry proved to be efficient in introducing functionalities and chemical crosslinks in the PGl chain and changing its thermal and mechanical properties

Intrinsically re-curable photopolymers containing dynamic thiol-Michael bonds

[Image: see text] The development of photopolymers that can be depolymerized and subsequently re-cured using the same light stimulus presents a significant technical challenge. A bio-sourced terpenoid structure, l-carvone, inspired the creation of a re-curable photopolymer in which the orthogonal reactivity of an irreversible thioether and a dynamic thiol-Michael bond enables both photopolymerization and thermally driven depolymerization of mechanically robust polymer networks. The di-alkene containing l-carvone was partially reacted with a multi-arm thiol to generate a non-crosslinked telechelic photopolymer. Upon further UV exposure, the photopolymer crosslinked into a mechanically robust network featuring reversible Michael bonds at junction points that could be activated to revert, or depolymerize, the network into a viscous telechelic photopolymer. The regenerated photopolymer displayed intrinsic re-curability over two recycles while maintaining the desirable thermomechanical properties of a conventional network: insolubility, resistance to stress relaxation, and structural integrity up to 170 °C. Our findings present an on-demand, re-curable photopolymer platform based on a sustainable feedstock

A renewably sourced, circular photopolymer resin for additive manufacturing

The additive manufacturing of photopolymer resins by means of vat photopolymerization enables the rapid fabrication of bespoke 3D-printed parts. Advances in methodology have continually improved resolution and manufacturing speed, yet both the process design and resin technology have remained largely consistent since its inception in the 1980s1. Liquid resin formulations, which are composed of reactive monomers and/or oligomers containing (meth)acrylates and epoxides, rapidly photopolymerize to create crosslinked polymer networks on exposure to a light stimulus in the presence of a photoinitiator2. These resin components are mostly obtained from petroleum feedstocks, although recent progress has been made through the derivatization of renewable biomass3,4,5,6 and the introduction of hydrolytically degradable bonds7,8,9. However, the resulting materials are still akin to conventional crosslinked rubbers and thermosets, thus limiting the recyclability of printed parts. At present, no existing photopolymer resin can be depolymerized and directly re-used in a circular, closed-loop pathway. Here we describe a photopolymer resin platform derived entirely from renewable lipoates that can be 3D-printed into high-resolution parts, efficiently deconstructed and subsequently reprinted in a circular manner. Previous inefficiencies with methods using internal dynamic covalent bonds10,11,12,13,14,15,16,17 to recycle and reprint 3D-printed photopolymers are resolved by exchanging conventional (meth)acrylates for dynamic cyclic disulfide species in lipoates. The lipoate resin platform is highly modular, whereby the composition and network architecture can be tuned to access printed materials with varied thermal and mechanical properties that are comparable to several commercial acrylic resins

Crosslinking of Electrospun Fibres from Unsaturated Polyesters by Bis-Triazolinediones (TAD)

Crosslinking of an unsaturated aliphatic polyester poly(globalide) (PGl) by bistriazolinediones (bisTADs) is reported. First, a monofunctional model compound, phenyl–TAD (PTAD), was tested for PGl functionalisation. 1H-NMR showed that PTAD–ene reaction was highly efficient with conversions up to 97%. Subsequently, hexamethylene bisTAD (HM–bisTAD) and methylene diphenyl bisTAD (MDP–bisTAD) were used to crosslink electrospun PGl fibres via one- and two-step approaches. In the one-step approach, PGl fibres were collected in a bisTAD solution for in situ crosslinking, which resulted in incomplete crosslinking. In the two-step approach, a light crosslinking of fibres was first achieved in a PGl non-solvent. Subsequent incubation in a fibre swelling bisTAD solution resulted in fully amorphous crosslinked fibres. SEM analysis revealed that the fibres’ morphology was uncompromised by the crosslinking. A significant increase of tensile strength from 0.3 ± 0.08 MPa to 2.7 ± 0.8 MPa and 3.9 ± 0.5 MPa was observed when PGI fibres were crosslinked by HM–bisTAD and MDP–bisTAD, respectively. The reported methodology allows the design of electrospun fibres from biocompatible polyesters and the modulation of their mechanical and thermal properties. It also opens future opportunities for drug delivery applications by selected drug loading