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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360901

RESUMO

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.


Assuntos
Biocatálise , Carbono/química , Carbono/farmacologia , Nitrogênio/química , Nitrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Coroa de Proteína/metabolismo , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Células A549 , Animais , Apirase/química , Apirase/farmacologia , Catalase/química , Catalase/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HeLa , Humanos , Ratos , Espécies Reativas de Oxigênio/metabolismo , beta-Galactosidase/química , beta-Galactosidase/farmacologia
2.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752

RESUMO

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.


Assuntos
Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
3.
Int J Biol Macromol ; 185: 966-982, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34237367

RESUMO

Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system.


Assuntos
Asparaginase/farmacologia , Quitosana/química , Polietilenoglicóis/química , Álcool de Polivinil/química , Asparaginase/química , Estabilidade de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Polietilenoglicóis/farmacologia , Stents , Temperatura , Molhabilidade
4.
ACS Appl Mater Interfaces ; 13(2): 2179-2188, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33405501

RESUMO

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.


Assuntos
Hidrolases de Éster Carboxílico/farmacologia , Erwinia amylovora/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Nanosferas/química , Doenças das Plantas/prevenção & controle , Percepção de Quorum/efeitos dos fármacos , Acil-Butirolactonas/metabolismo , Hidrolases de Éster Carboxílico/administração & dosagem , Hidrolases de Éster Carboxílico/genética , Evolução Molecular Direcionada/métodos , Enzimas Imobilizadas/administração & dosagem , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/farmacologia , Erwinia amylovora/fisiologia , Modelos Moleculares , Peptídeos/química , Doenças das Plantas/microbiologia , Pyrus/microbiologia
5.
Carbohydr Polym ; 252: 117138, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33183597

RESUMO

Bacterial adhesion infection caused by medical materials in clinical application has become a serious threat, and it urgently needs new strategies to deal with these clinical challenges. In this work, LED209, a highly selective histidine sensor kinase inhibitor of Gram-negative bacteria, was covalently attached on cellulose membrane (CM) via click reaction. The data of contact angle measurements, ATR-FTIR and X-ray photoelectron spectroscopy confirmed the successful synthesis of LED-CM. In addition, the results of antibacterial activity of the membranes shown that LED-CM exhibited excellent anti-adhesion ability to Enterohemorrhagic Escherichia coli (EHEC), and significantly reduced the formation of bacterial biofilm. Importantly, LED-CM was able to repress the expression of virulence genes in EHEC. Furthermore, LED209-functionalized cellulose membrane indicated no cytotoxicity to mammalian cells. Hence, our present work demonstrated that CM modified with LED209 possessed markedly anti-adhesion activity against EHEC, which offered a potent antimicrobial material for combating bacterial infections.


Assuntos
Proteínas da Membrana Bacteriana Externa/antagonistas & inibidores , Enzimas Imobilizadas/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli O157/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Complexos Multienzimáticos/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Celulose/química , Membranas Artificiais , Camundongos , Células NIH 3T3
6.
Int J Biol Macromol ; 165(Pt B): 3065-3077, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33127544

RESUMO

We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.


Assuntos
Celulose Oxidada/química , Papaína/química , Peptídeo Hidrolases/química , Pele/efeitos dos fármacos , Acetobacteraceae/enzimologia , Adsorção/efeitos dos fármacos , Celulose Oxidada/farmacologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Concentração de Íons de Hidrogênio , Oxirredução , Papaína/biossíntese , Papaína/farmacologia , Peptídeo Hidrolases/farmacologia , Pele/lesões
7.
Carbohydr Polym ; 246: 116625, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32747262

RESUMO

Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of biofilms can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35 ±â€¯1.05 mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic reduction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.


Assuntos
Acetobacteraceae/química , Bandagens , Biofilmes/efeitos dos fármacos , Celulose/química , Glicosídeo Hidrolases/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Acetobacteraceae/fisiologia , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Celulose/biossíntese , Celulose/isolamento & purificação , Clonagem Molecular , Enzimas Imobilizadas/biossíntese , Enzimas Imobilizadas/genética , Enzimas Imobilizadas/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/genética , Camundongos , Domínios Proteicos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
8.
J Mater Chem B ; 8(20): 4395-4401, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32400814

RESUMO

We developed a polymer-encapsulated DNase, n(DNase), which can efficiently accumulate in biofilm and expose the DNase to cleave the eDNA of the biofilm. CLSM and crystal violet staining results demonstrated effective biofilm disintegration (92.2%) when treated with n(DNase). This work demonstrated a general approach for coating matrix-dispersion enzymes to achieve biofilm disintegration and provided a promising strategy for treating biofilm-associated infections.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Desoxirribonucleases/farmacologia , Enzimas Imobilizadas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/administração & dosagem , Desoxirribonucleases/administração & dosagem , Portadores de Fármacos/química , Sinergismo Farmacológico , Enzimas Imobilizadas/administração & dosagem , Humanos , Polímeros/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia
9.
ACS Appl Mater Interfaces ; 12(23): 25625-25632, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32383848

RESUMO

The prolonged use of enzymes under oxidative stress is a major challenge in enabling effective enzymatic reaction pathways. Herein, we report a biomimetic antioxidant defensive strategy capable of providing adequate protection of enzymes against superoxide-mediated oxidation. Superoxide dismutase (SOD) and catalase (CAT) were chosen as scavengers and covalently encapsulated into silica nanoreactors, together with glucose dehydrogenase (GDH), which simultaneously should produce the coenzyme nicotinamide adenine dinucleotide (NADH, reduced form). By the enzymatic reactions of SOD and CAT, the interior of silica nanoreactors becomes a "ROS safe zone" to protect the glucose-dependent NADH production of coencapsulated GDH. We further combined this protected NADH-producing module with photocatalytic nanoparticles that enable the light-triggered oxidation of NADH back to NAD+ (oxidized form). In combination, these two modules allow interconversion between NAD+ and NADH by the addition of glucose or by light irradiation (LED lamp or sunlight). This protection and regeneration strategy is a versatile tool for enzyme applications for biological reactors, catalysis, or prototypes of artificial organelles or building blocks that contains fragile biomolecules and rely on the coenzyme NAD+/NADH.


Assuntos
Catalase/farmacologia , Enzimas Imobilizadas/farmacologia , Glucose 1-Desidrogenase/farmacologia , NAD/metabolismo , Nanopartículas/química , Superóxido Dismutase/farmacologia , Biomimética/métodos , Catalase/química , Linhagem Celular Tumoral , Enzimas Imobilizadas/química , Glucose/química , Glucose/metabolismo , Glucose 1-Desidrogenase/química , Humanos , Luz , NAD/química , Nanopartículas/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Polímeros/química , Polímeros/efeitos da radiação , Dióxido de Silício/química , Superóxido Dismutase/química , Superóxidos/química , Superóxidos/metabolismo
10.
Angew Chem Int Ed Engl ; 59(31): 12698-12702, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32297692

RESUMO

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.


Assuntos
Antibacterianos/farmacologia , DNA/química , Portadores de Fármacos/química , Muramidase/farmacologia , Nanoestruturas/química , Animais , Antibacterianos/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/toxicidade , Bacillus subtilis/química , Bacillus subtilis/efeitos dos fármacos , Células COS , Chlorocebus aethiops , DNA/toxicidade , Portadores de Fármacos/toxicidade , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Muramidase/química , Nanoestruturas/toxicidade , Conformação de Ácido Nucleico
11.
Methods Mol Biol ; 2118: 213-225, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32152982

RESUMO

Direct immobilization of functional proteins on gold nanoparticles (AuNPs) affects their structure and function. Changes may vary widely and range from strong inhibition to the enhancement of protein function. More often though the outcome of direct protein immobilization results in protein misfolding and the loss of protein activity. Additional complications arise when the protein being immobilized is a zymogen which requires and relies on additional protein-protein interactions to exert its function. Here we describe molecular design of a glutathione-S-transferase-Staphylokinase fusion protein (GST-SAK) and its conjugation to AuNPs. The multivalent AuNP-(GST-SAK)n complexes generated show plasminogen activation activity in vitro. The methods described are transferable and could be adapted for conjugation and functional analysis of other plasminogen activators, thrombolytic preparations or other functional enzymes.


Assuntos
Glutationa Transferase/genética , Ouro/química , Metaloendopeptidases/genética , Ativadores de Plasminogênio/farmacologia , Proteínas Recombinantes/farmacologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Fibrinogênio/metabolismo , Glutationa Transferase/química , Humanos , Nanopartículas Metálicas , Metaloendopeptidases/química , Modelos Moleculares , Ativadores de Plasminogênio/química , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química
12.
Int J Mol Sci ; 21(5)2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32121010

RESUMO

The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. Lysozyme degrades bacterial cell wall without involving antibiotic resistance and has become a new antibacterial strategy. However, direct use of native, active proteins in clinical settings is not practical as it is fragile under various conditions. In this study, lysozyme was integrated into chitosan nanoparticles (CS-NPs) by the ionic gelation technique to obtain lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs) and then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), which showed a small particle size (243.1 ± 2.1 nm) and positive zeta potential (22.8 ± 0.2 mV). The immobilization significantly enhanced the thermal stability and reusability of lysozyme. In addition, compared with free lysozyme, Lys-CS-NPs exhibited superb antibacterial properties according to the results of killing kinetics in vitro and measurement of the minimum inhibitory concentration (MIC) of CS-NPs and Lys-CS-NPs against Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). These results suggest that the integration of lysozyme into CS-NPs will create opportunities for the further potential applications of lysozyme as an anti-bacterium agent.


Assuntos
Antibacterianos/farmacologia , Quitosana/química , Portadores de Fármacos/química , Muramidase/farmacologia , Nanopartículas/química , Difusão Dinâmica da Luz , Estabilidade Enzimática , Enzimas Imobilizadas/farmacologia , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Temperatura
13.
ACS Appl Mater Interfaces ; 12(19): 21311-21321, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32023023

RESUMO

Bacterial pathogens are responsible for millions of cases of illnesses and deaths each year throughout the world. The development of novel surfaces and coatings that effectively inhibit and prevent bacterial attachment, proliferation, and growth is one of the crucial steps for tackling this global challenge. Herein, we report a dual-functional coating for aluminum surfaces that relies on the controlled immobilization of lysozyme enzyme (muramidase) into interstitial spaces of presintered, nanostructured thin film based on ∼200 nm silica nanoparticles and the sequential chemisorption of an organofluorosilane to the available interfacial areas. The mean diameter of the resultant lysozyme microdomains was 3.1 ± 2.5 µm with an average spacing of 8.01 ± 6.8 µm, leading to a surface coverage of 15.32%. The coating had an overall root-mean-square (rms) roughness of 539 ± 137 nm and roughness factor of 1.50 ± 0.1, and demonstrated static, advancing, and receding water contact angles of 159.0 ± 1.0°, 155.4 ± 0.6°, and 154.4 ± 0.6°, respectively. Compared to the planar aluminum, the coated surfaces produced a 6.5 ± 0.1 (>99.99997%) and 4.0 ± 0.1 (>99.99%) log-cycle reductions in bacterial surfaces colonization against Gram-negative Salmonella Typhimurium LT2 and Gram-positive Listeria innocua, respectively. We anticipate that the implementation of such a coating strategy on healthcare environments and surfaces and food-contact surfaces can significantly reduce or eliminate potential risks associated with various contamination and cross-contamination scenarios.


Assuntos
Antibacterianos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Desinfetantes/farmacologia , Enzimas Imobilizadas/farmacologia , Muramidase/farmacologia , Alumínio/química , Antibacterianos/química , Desinfetantes/química , Enzimas Imobilizadas/química , Interações Hidrofóbicas e Hidrofílicas , Listeria/efeitos dos fármacos , Muramidase/química , Nanopartículas/química , Salmonella typhimurium/efeitos dos fármacos , Dióxido de Silício/química , Molhabilidade
14.
Methods Mol Biol ; 2089: 235-243, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31773658

RESUMO

Ligand fishing is a convenient bioanalytical screening method that is based on the affinity selection of a ligand from a complex biological sample by an immobilized receptor. It is a versatile affinity-based screening approach and it has found application in multiple interacting pairs such as enzyme-inhibitor/activator, antigen-antibody, receptor-ligand, and protein-protein. Important parameters that affect the successful operation of the method are the high specificity and strong binding affinity of the interacting pair (e.g., enzyme-ligand complex) and the elution of the bound ligand from the complex. This chapter provides protocols for the synthesis of affinity adsorbent and its application in off-line ligand-fishing procedure for a 6His-tagged glutathione transferase (GST).


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Anticorpos/metabolismo , Cromatografia de Afinidade/métodos , Descoberta de Drogas/métodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Glutationa Transferase/metabolismo , Ligantes , Ligação Proteica/fisiologia , Proteínas/metabolismo
15.
Nanomedicine (Lond) ; 15(1): 23-39, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868116

RESUMO

Aim: Superoxide dismutase (SOD) and catalase (CAT) immobilized on gold nanoparticles (AuNP) and silver nanoparticles (AgNP) nanoparticles were used to reduce UV radiation-induced oxidative stress in rat skin. Materials & methods: The antioxidant influence of the enzymes was investigated on level of malondialdehyde, 8-hydroksy-2'deoksyguanozine, myeloperoxidase, total antioxidant capacity, SOD2 and CAT activity and expression, and glutathione and glutathione peroxidase activity. Results: The application of immobilized SOD and CAT on UV-irradiated skin reduced malondialdehyde and 8-hydroksy-2'deoksyguanozine levels also SOD and CAT activity and expression increased. The tested enzymes influence glutathione peroxidase activity and level of total antioxidant capacity and glutathione. Conclusion: Immobilized enzymes increased the antioxidative potential of skin following UV irradiation.


Assuntos
Antioxidantes/farmacologia , Enzimas Imobilizadas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Pele/efeitos dos fármacos , Animais , Antioxidantes/química , Catalase/química , Catalase/farmacologia , Enzimas Imobilizadas/química , Glutationa/química , Ouro/química , Humanos , Malondialdeído/química , Nanopartículas Metálicas/química , Protetores contra Radiação/química , Ratos , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Pele/efeitos da radiação , Superóxido Dismutase/química , Superóxido Dismutase/farmacologia , Raios Ultravioleta/efeitos adversos
16.
Recent Pat Biotechnol ; 14(2): 154-163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31724520

RESUMO

BACKGROUND: L-asparaginase (L-AsnA) enzyme has gained significant attention in the food, biocatalysts and pharmaceutics industry. It (L-AsnA) has been widely used in food processing industries as a promising acrylamide mitigating agent and as a therapeutic agent in the treatment of certain human cancers. OBJECTIVE: Based on US Patent (4,433,054; 1984), L-asparaginase (L-AsnA) enzyme is immobilized by admixing the active enzyme on the polysaccharide to be in a gel form. The storage stability of immobilized L-AsnA enzyme and its anti-proliferation and antiviral activity were determined. METHODS: In the present study, S. maxima was cultured at large scales (300 liter) for the production of enough extracellular L-asparaginase (L-AsnA) using modified (high N concentration) Zarrouk medium as we reported in a previous study. L-AsnA was immobilized on natural polymers, as agar cake beads, agarose pieces and gelatin blocks, in order to evaluate the efficiency of physical entrapment techniques. Anti-proliferation properties of L-AsnA against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines were assessed by the MTT cell viability method. In addition, the antiviral activity against Coxsackie B3 (CSB3) Virus was assessed. RESULTS: The highest L-AsnA immobilized activity and immobilization yield were achieved with agar cakes bead. The purified S. maxima L-AsnA showed good antiviral activity against Coxsackie B3 (CSB3) Virus in a dose-dependent manner with an IC50 value 17.03 µg/ml. The antiviral mode of action is presumably due to their capability of inhibiting attachment, blocking the adsorption and penetration event of the viral replication cycle with 89.24%, 72.78% and 72.78%, respectively. Also, S. maxima L-AsnA showed anti-proliferation effect against lung carcinoma A549, hepatocellular carcinoma Hep-G2 and prostate carcinoma PC3 human cancer cell lines, with an IC50 of 22.54, 24.65 and 56.61 µg/ml, respectively. CONCLUSION: It is interesting to favor L-asparaginase of S. maxima which showed antiviral activity and anti-proliferation effect against different types of human cell lines. Thus, S. maxima microalgae might be a good source for L-AsnA enzymes and can be immobilized on natural polymers.


Assuntos
Antivirais , Asparaginase , Proteínas de Bactérias , Enzimas Imobilizadas , Spirulina/enzimologia , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Asparaginase/química , Asparaginase/metabolismo , Asparaginase/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Enterovirus Humano B , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacologia , Células Hep G2 , Humanos , Células PC-3 , Patentes como Assunto
17.
Fungal Biol ; 123(12): 875-886, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31733730

RESUMO

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a glycoprotein having many biotechnological applications. In the present study, CDHs isolated from Phlebia lindtneri (PlCDH), Phanerochaete chrysosporium (PchCDH), Cerrena unicolor (CuCDH), and Pycnoporus sanguineus (PsCDH) were studied the first time for their ability to generate antioxidant and antimicrobial agents. The aim of the research was to evaluate the antioxidant and antimicrobial activity of systems composed of four CDHs and lactose or cellobiose as a reaction substrate. The free radical scavenging effect of free and immobilised enzymes was evaluated using the DPPH method. The lowest values of EC50 (10.04 ± 0.75 µg/ml) was noted for PlCDH/lactose and for PlCDH/cellobiose (12.06 ± 1.35 µg/ml). The EC50value reached 12.6 ± 1.51 µg/ml in the case of PsCDH/lactose and 15.96 ± 1.35 for PsCDH. The CDH preparations were also effectively immobilised in alginate (the immobilisation efficiency expressed as a protein yield ranged from 61.6 to 100 %). The operational stability expressed as a scavenging effect showed the possibility of using the alginate beads 4 times. Both the free and immobilised CDHs as well as the CDH/substrate were tested against Gram-negative Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Gram-positive Staphylococcus aureus ATCC 25923 bacteria. All samples, except PlCDH, were potentially effective in suppression of bacterial growth. The highest percentage of inhibition (100 %) was obtained for S. aureus bacteria using PsCDH and PchCDH with lactose as a substrate, whereas a slightly lesser effect was observed for E. coli and P. aeruginosa bacterial cells, i.e. 64.1 % and 86.5 % (PsCDH) and 94.1 % and 41.4 % (PchCDH), respectively. Furthermore, the concentrations of the reaction products (aldonic acids and hydrogen peroxide) were quantified and the surface morphology of the alginate beads was analysed using SEM visualisation.


Assuntos
Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Basidiomycota/enzimologia , Desidrogenases de Carboidrato/isolamento & purificação , Desidrogenases de Carboidrato/farmacologia , Basidiomycota/isolamento & purificação , Compostos de Bifenilo/metabolismo , Desidrogenases de Carboidrato/metabolismo , Celobiose/metabolismo , Enzimas Imobilizadas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Lactose/metabolismo , Testes de Sensibilidade Microbiana , Picratos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Madeira/microbiologia
18.
Sci Rep ; 9(1): 15730, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673063

RESUMO

Robust immobilization of glucose oxidase (GOx) enzyme was achieved on poly(ethylene terephthalate) nonwoven fabric (PN) after integration of favourable surface functional groups through plasma treatments [atmospheric pressure-AP or cold remote plasma-CRP (N2 + O2)] and/or chemical grafting of hyperbranched dendrimers [poly-(ethylene glycol)-OH or poly-(amidoamine)]. Absorption, stability, catalytic behavior of immobilized enzymes and reusability of resultant fibrous bio-catalysts were comparatively studied. Full characterization of PN before and after respective modifications was carried out by various analytical, instrumental and arithmetic techniques. Results showed that modified polyester having amine terminal functional groups pledged better surface property providing up to 31% enzyme loading, and 81% active immobilized enzymes. The activity of the enzyme was measured in terms of interaction aptitude of GOx in a given time to produce hydrogen peroxide using colorimetric assay. The immobilized GOx retained 50% of its original activity after being reused six (06) times and exhibited improved stability compared with the free enzyme in relation to temperature. The reaction kinetics, loading efficiency, leaching, and reusability analysis of enzyme allowed drawing a parallel to the type of organic moiety integrated during GOx immobilization. In addition, resultant fibrous bio-catalysts showed substantial antibacterial activity against pathogenic bacteria strains (Staphylococcus epidermidis and Escherichia coli) in the presence of oxygen and glucose. These results are of great importance because they provide proof-of-concept for robust immobilization of enzymes on surface-modified fibrous polyester fabric for potential bio-industrial applications.


Assuntos
Dendrímeros/química , Glucose Oxidase/química , Gases em Plasma/química , Poliésteres/química , Biocatálise , Colorimetria , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/farmacologia , Escherichia coli/efeitos dos fármacos , Glucose Oxidase/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Staphylococcus epidermidis/efeitos dos fármacos
19.
ACS Appl Mater Interfaces ; 11(43): 39436-39448, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31580644

RESUMO

Laccases are multi-copper oxidase enzymes having widespread applications in various biotechnological fields. However, low stability of free enzymes restricts their industrial use. Development of effective methods to preserve and even increase the enzymatic activity is critical to maximize their use, though this remains a challenge. In the present study we immobilized Trametes versicolor laccase on pH-responsive (and charge-switchable) Pluronic-stabilized silver nanoparticles (AgNPsTrp). Our results demonstrate that colloidal stabilization of AgNPsTrp with the amphiphilic copolymer Pluronic F127 enhances enzyme activity (AgNPsTrpF1 + Lac6) by changing the active site microenvironment, which is confirmed by circular dichroism (CD) and fluorescence spectroscopy. Detailed kinetic and thermodynamic studies reveal a facile strategy to improve the protein quality by lowering the activation energy and expanding the temperature window for substrate hydrolysis. The immobilized nanocomposite did not show any change in flow behavior which indirectly suggests that the enzyme stability is maintained, and the enzyme did not aggregate or unfold upon immobilization. Finally, assessing the anticancer efficacy of this nanocomposite in breast cancer MCF-7 cells shows the inhibition of cell proliferation through ß-estradiol degradation and cells apoptosis. To understand the molecular mechanism involved in this process, semi qRT-PCR experiments were performed, which indicated significant decrease in the mRNA levels of anti-apoptotic genes, for example, BCL-2 and NF-kß, and increase in the mRNA level of pro-apoptotic genes like p53 in treated cells, compared to control. Overall, this study offers a completely new strategy for tailoring nano-bio-interfaces with improved activity and stability of laccase.


Assuntos
Neoplasias da Mama , Enzimas Imobilizadas , Proteínas Fúngicas , Lacase , Poloxâmero , Polyporaceae/enzimologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lacase/química , Lacase/farmacologia , Células MCF-7 , Proteínas de Neoplasias/biossíntese , Poloxâmero/química , Poloxâmero/farmacologia
20.
ACS Appl Mater Interfaces ; 11(46): 42917-42931, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31635454

RESUMO

The unique characteristics of a tumor microenvironment (TME) enable the development of new tumor-therapeutic modalities with high efficiency, biosafety, and tumor specificity. In this work, we report on the construction of photothermal-enhanced and nanocatalyst-enabled sequential catalytic reaction for TME-specific cancer therapy. This conceptual advance is achieved by engineering the surface of two-dimensional Ti3C2 MXene with two separate catalysts, including natural glucose oxidase (GOD) as glucose catalysts and superparamagnetic iron oxide nanoparticles (IONPs) as Fenton-reaction nanocatalysts. A sequential catalytic reaction is triggered by using GOD for catalyzing the tumor-overtaken glucose to generate large amounts of hydrogen peroxide molecules. Subsequently IONPs can catalyze the transformation of pregenerated hydrogen peroxide into large amounts of highly toxic hydroxyl radicals to kill the cancer cells subsequently in TME-enabled acidity condition. The two-dimensional (2D) Ti3C2 MXene matrix efficiently converts the near-infrared light into thermal energy to synergistically enhance the catalytic efficiency of this sequential catalytic reaction and therefore achieve the high synergistic cancer-therapeutic outcome, accompanied with the high biocompatibility of the constructed composite nanocatalysts. Both in vitro cancer-cell evaluation and in vivo tumor xenograft on nude mice with complete tumor eradication demonstrate the high synergistic efficiency of photothermal-enhanced sequential nanocatalytic cancer therapy. Therefore, this work substantially broadens the biomedical applications of 2D MXenes to nanocatalytic cancer therapy by enhancing the Fenton reaction-based nanocatalytic therapy via converting the near-infrared light into thermal energy and subsequently elevating the local Fenton-reaction temperature.


Assuntos
Enzimas Imobilizadas , Glucose Oxidase , Hipertermia Induzida , Nanopartículas de Magnetita , Neoplasias Experimentais , Fototerapia , Titânio , Microambiente Tumoral/efeitos dos fármacos , Animais , Catálise , Linhagem Celular Tumoral , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Feminino , Glucose Oxidase/química , Glucose Oxidase/farmacologia , Peróxido de Hidrogênio/metabolismo , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Titânio/química , Titânio/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
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