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1.
Metab Eng ; 57: 162-173, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726216

RESUMO

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

2.
Biomolecules ; 9(12)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31816991

RESUMO

Bladder cancer is the ninth most common cancer worldwide. Due to a high risk of recurrence and progression of bladder cancer, every patient needs long-term surveillance, which includes regular cystoscopy, sometimes followed by a biopsy of suspicious lesions or resections of recurring tumours. This study addresses the development of novel biohybrid nanocomplexes representing upconversion nanoparticles (UCNP) coupled to antibodies for photoluminescent (PL) detection of bladder cancer cells. Carrying specific antibodies, these nanoconjugates selectively bind to urothelial carcinoma cells and make them visible by emitting visible PL upon excitation with deeply penetrating near-infrared light. UCNP were coated with a silica layer and linked to anti-Glypican-1 antibody MIL38 via silica-specific solid-binding peptide. Conjugates have been shown to specifically attach to urothelial carcinoma cells with high expression of Glypican-1. This result highlights the potential of produced conjugates and conjugation technology for further studies of their application in the tumour detection and fluorescence-guided resection.

3.
Biomolecules ; 10(1)2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31861313

RESUMO

Linker-protein G (LPG) is a bifunctional fusion protein composed of a solid-binding peptide (SBP, referred as the "linker") with high affinity to silica-based compounds and a Streptococcus protein G (PG), which binds antibodies. The binding mechanisms of LPG to silica-based materials was studied using different biophysical techniques and compared to that of PG without the linker. LPG displayed high binding affinity to a silica surface (KD = 34.77 ± 11.8 nM), with a vertical orientation, in comparison to parent PG, which exhibited no measurable binding affinity. Incorporation of the linker in the fusion protein, LPG, had no effect on the antibody-binding function of PG, which retained its secondary structure and displayed no alteration of its chemical stability. The LPG system provided a milder, easier, and faster affinity-driven immobilization of antibodies to inorganic surfaces when compared to traditional chemical coupling techniques.

4.
Nanomaterials (Basel) ; 9(9)2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527483

RESUMO

In recent years, it has become apparent that cancer nanomedicine's reliance on synthetic nanoparticles as drug delivery systems has resulted in limited clinical outcomes. This is mostly due to a poor understanding of their "bio-nano" interactions. Protein-based nanoparticles (PNPs) are rapidly emerging as versatile vehicles for the delivery of therapeutic and diagnostic agents, offering a potential alternative to synthetic nanoparticles. PNPs are abundant in nature, genetically and chemically modifiable, monodisperse, biocompatible, and biodegradable. To harness their full clinical potential, it is important for PNPs to be accurately designed and engineered. In this review, we outline the recent advancements and applications of PNPs in cancer nanomedicine. We also discuss the future directions for PNP research and what challenges must be overcome to ensure their translation into the clinic.

5.
Artigo em Inglês | MEDLINE | ID: mdl-31157220

RESUMO

In recent years, the versatile phototrophic protist Euglena gracilis has emerged as an interesting candidate for application-driven research and commercialisation, as it is an excellent source of dietary protein, pro(vitamins), lipids, and the ß-1,3-glucan paramylon only found in euglenoids. From these, paramylon is already marketed as an immunostimulatory agent in nutraceuticals. Bioproducts from E. gracilis can be produced under various cultivation conditions discussed in this review, and their yields are relatively high when compared with those achieved in microalgal systems. Future challenges include achieving the economy of large-scale cultivation. Recent insights into the complex metabolism of E. gracilis have highlighted unique metabolic pathways, which could provide new leads for product enhancement by genetic modification of the organism. Also, development of molecular tools for strain improvement are emerging rapidly, making E. gracilis a noteworthy challenger for microalgae such as Chlorella spp. and their products currently on the market.

6.
N Biotechnol ; 52: 9-18, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30954671

RESUMO

The interactions between biomolecules and solid surfaces play an important role in designing new materials and applications which mimic nature. Recently, solid-binding peptides (SBPs) have emerged as potential molecular building blocks in nanobiotechnology. SBPs exhibit high selectivity and binding affinity towards a wide range of inorganic and organic materials. Although these peptides have been widely used in various applications, there is a need to understand the interaction mechanism between the peptide and its material substrate, which is challenging both experimentally and theoretically. This review describes the main characterisation techniques currently available to study SBP-surface interactions and their contribution to gain a better insight for designing new peptides for tailored binding.


Assuntos
Modelos Moleculares , Peptídeos/metabolismo , Sequência de Aminoácidos , Fenômenos Biofísicos , Peptídeos/química , Ligação Proteica
7.
Anal Chim Acta ; 1066: 136-145, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31027529

RESUMO

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.


Assuntos
Aldeído Oxirredutases/metabolismo , Biocatálise , Cromatografia Líquida/métodos , Ácido Glucárico/metabolismo , Configuração de Carboidratos , Escherichia coli/enzimologia , Ácido Glucárico/química , Rhizobiaceae/enzimologia
8.
J Ind Microbiol Biotechnol ; 46(6): 769-781, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30806871

RESUMO

Enzymatic degradation of the ß-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-ß-D-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis ß-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.


Assuntos
Euglena gracilis/enzimologia , Citometria de Fluxo/métodos , Glucanos/análise , Escherichia coli/metabolismo , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Hidrólise , Proteômica
9.
Biotechnol Adv ; 37(1): 91-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30521853

RESUMO

Single enzyme systems or engineered microbial hosts have been used for decades but the notion of assembling multiple enzymes into cell-free synthetic pathways is a relatively new development. The extensive possibilities that stem from this synthetic concept makes it a fast growing and potentially high impact field for biomanufacturing fine and platform chemicals, pharmaceuticals and biofuels. However, the translation of individual single enzymatic reactions into cell-free multi-enzyme pathways is not trivial. In reality, the kinetics of an enzyme pathway can be very inadequate and the production of multiple enzymes can impose a great burden on the economics of the process. We examine here strategies for designing synthetic pathways and draw attention to the requirements of substrates, enzymes and cofactor regeneration systems for improving the effectiveness and sustainability of cell-free biocatalysis. In addition, we comment on methods for the immobilisation of members of a multi-enzyme pathway to enhance the viability of the system. Finally, we focus on the recent development of integrative tools such as in silico pathway modelling and high throughput flux analysis with the aim of reinforcing their indispensable role in the future of cell-free biocatalytic pathways for biomanufacturing.


Assuntos
Sistema Livre de Células/metabolismo , Enzimas/metabolismo , Engenharia Metabólica/tendências , Biologia Sintética , Biocatálise , Sistema Livre de Células/química , Simulação por Computador , Enzimas/química , Enzimas/genética , Humanos , Cinética , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo
10.
Genes (Basel) ; 9(7)2018 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-30041491

RESUMO

In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP's intrinsic properties and to impart them with novel functionalities.

11.
Carbohydr Polym ; 196: 339-347, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29891305

RESUMO

A hydrothermal microwave pretreatment was established to facilitate the enzymatic production of soluble bioactive ß-1,3-glucans from the recalcitrant substrate paramylon. The efficacy of this pretreatment was monitored with a newly developed direct Congo Red dye-based assay over a range of temperatures. Microwave pretreatment at 170 °C for 2 min resulted in a significantly enhanced enzymatic hydrolysis of paramylon. The action of endo-ß-1,3- and exo- ß-1,3-glucanases on the microwave-pretreated paramylon produced soluble ß-1,3-glucans with degrees of polymerisation (DP) ranging from 2-59 and 2-7, respectively. In comparison, acid-mediated hydrolysis of untreated paramylon resulted in ß-1,3-glucans with a DP range of 2-38. The hydrolysates were assayed on their immunostimulatory effect on murine macrophages by measuring the production of the inflammation-linked marker tumour necrosis factor alpha (TNFα) using immunofluorescence. All of the tested hydrolysis products were shown to induce TNFα production, with the most significant immunostimulatory effect observed with the hydrolysate from the exo-ß-1,3-glucanase treatment.


Assuntos
Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/farmacologia , Enzimas/metabolismo , Glucanos/química , Micro-Ondas , beta-Glucanas/síntese química , beta-Glucanas/farmacologia , Adjuvantes Imunológicos/química , Animais , Linhagem Celular , Técnicas de Química Sintética , Hidrólise , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Solubilidade , Temperatura de Transição , beta-Glucanas/química
12.
ACS Appl Mater Interfaces ; 10(20): 17437-17447, 2018 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-29701945

RESUMO

Nanoparticle surface engineering can change its chemical identity to enable surface coupling with functional biomolecules. However, common surface coupling methods such as physical adsorption or chemical conjugation often suffer from the low coupling yield, poorly controllable orientation of biomolecules, and steric hindrance during target binding. These issues limit the application scope of nanostructures for theranostics and personalized medicine. To address these shortfalls, we developed a rapid and versatile method of nanoparticle biomodification. The method is based on a SiO2-binding peptide that binds to the nanoparticle surface and a protein adaptor system, Barnase*Barstar protein pair, serving as a "molecular glue" between the peptide and the attached biomolecule. The biomodification procedure shortens to several minutes, preserves the orientation and functions of biomolecules, and enables control over the number and ratio of attached molecules. The capabilities of the proposed biomodification platform were demonstrated by coupling different types of nanoparticles with DARPin9.29 and 4D5scFv-molecules that recognize the human epidermal growth factor receptor 2 (HER2/neu) oncomarker-and by subsequent highly selective immunotargeting of the modified nanoparticles to different HER2/neu-overexpressing cancer cells in one-step or two-step (by pretargeting with HER2/neu-recognizing molecule) modes. The method preserved the biological activity of the DARPin9.29 molecules attached to a nanoparticle, whereas the state-of-the-art carbodiimide 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N-hydroxysulfosuccinimide method of conjugation led to a complete loss of the functional activity of the DARPin9.29 nanoparticle-protein complex. Moreover, the method allowed surface design of nanoparticles that selectively interacted with antigens in complex biological fluids, such as whole blood. The demonstrated capabilities show this method to be a promising alternative to commonly used chemical conjugation techniques in nanobiotechnology, theranostics, and clinical applications.

13.
ACS Sens ; 3(2): 320-326, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29308890

RESUMO

We report an aggregation-induced emission fluorogen (AIEgen)-based turn-on fluorescent aptasensor able to detect the ultrasmall concentration of intracellular IFN-γ. The aptasensor consists of an IFN-γ aptamer labeled with a fluorogen with a typical aggregation-induced emission (AIE) characteristic, which shows strong red emission only in the presence of IFN-γ. The aptasensor is able to effectively monitor intracellular IFN-γ secretion with the lowest detection limit of 2 pg mL-1, and it is capable of localizing IFN-γ in live cells during secretion, with excellent cellular permeability and biocompatibility as well as low cytotoxicity. This probe is able to localize the intracellular IFN-γ at a low concentration <10 pg mL-1, and it is successfully used for real-time bioimaging. This simple and highly sensitive sensor may enable the exploration of cytokine pathways and their dynamic secretion process in the cellular environment. It provides a universal sensing platform for monitoring a spectrum of molecules secreted by cells.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Interferon gama/análise , Animais , Linhagem Celular , Camundongos , Sensibilidade e Especificidade
14.
Adv Exp Med Biol ; 1030: 21-36, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29081048

RESUMO

Some peptides are able to bind to inorganic materials such as silica and gold. Over the past decade, Solid-binding peptides (SBPs) have been used increasingly as molecular building blocks in nanobiotechnology. These peptides show selectivity and bind with high affinity to a diverse range of inorganic surfaces e.g. metals, metal oxides, metal compounds, magnetic materials, semiconductors, carbon materials, polymers and minerals. They can be used in applications such as protein purification and synthesis, assembly and the functionalization of nanomaterials. They offer simple and versatile bioconjugation methods that can increase biocompatibility and also direct the immobilization and orientation of nanoscale entities onto solid supports without impeding their functionality. SBPs have been employed in numerous nanobiotechnological applications such as the controlled synthesis of nanomaterials and nanostructures, formation of hybrid biomaterials, immobilization of functional proteins and improved nanomaterial biocompatibility. With advances in nanotechnology, a multitude of novel nanomaterials have been designed and synthesized for diagnostic and therapeutic applications. New approaches have been developed recently to exert a greater control over bioconjugation and eventually, over the optimal and functional display of biomolecules on the surfaces of many types of solid materials. In this chapter we describe SBPs and highlight some selected examples of their potential applications in biomedicine.


Assuntos
Materiais Biocompatíveis/química , Nanopartículas Metálicas/química , Nanotecnologia/métodos , Peptídeos/química , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Materiais Biocompatíveis/metabolismo , Metais/química , Metais/metabolismo , Óxidos/química , Óxidos/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Semicondutores , Propriedades de Superfície
15.
Chemosphere ; 184: 694-699, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28633064

RESUMO

Soils contaminated with mercury (Hg) have proved expensive and logistically difficult to remediate. Research continues into finding suitable environmentally-friendly and efficient ways of achieving this end. Bioremediation is an option, which employs the strategies microorganisms have evolved to deal with Hg. One microbial strategy involves uptake and intracellular volatilisation of mercuric ions, which passively diffuse from the cell and back into the atmosphere. In this work, Pseudomonas veronii cells grown to stationary phase were immobilised in a xanthan gum-based biopolymer via encapsulation. The P. veronii-biopolymer mix was then coated onto natural zeolite granules. Zeolite immobilised cells remained viable for at least 16 weeks stored under ambient room temperature. Furthermore, the immobilised cells were shown to retain both viability and Hg volatilisation functionality after transportation from Australia to the USA, where they were applied to Hg contaminated soil. Maximum flux rates exceeded 10 µg Hg m2 h-1 from mine tailings (≈7 mg kg-1 Hg with 50% v/v water). This was 4 orders of magnitude above background flux levels. It is envisioned that emitted gaseous elemental mercury (GEM) can be readily captured, and transformed back into metallic Hg, which can then be stored appropriately or recycled. This breaks the Hg cycle, as GEM is no longer translocated back to the atmospheric compartment. The immobilising excipients used in this research overcome many logistical issues with delivery of suitable microbial loads to locations of mercury contamination and presents a facile and inexpensive method of augmenting contaminated sites with selected microbial consortia for bioremediation.


Assuntos
Biodegradação Ambiental , Monitoramento Ambiental , Mercúrio/análise , Poluentes do Solo/análise , Atmosfera , Austrália , Mercúrio/metabolismo , Mineração , Solo , Microbiologia do Solo , Poluentes do Solo/metabolismo , Volatilização
16.
Nanoscale ; 9(15): 4934-4943, 2017 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-28368062

RESUMO

The detection of cytokines in body fluids, cells, tissues and organisms continues to attract considerable attention due to the importance of these key cell signalling molecules in biology and medicine. We report a graphene quantum dot (GQD) based aptasensor able to specifically detect ultra-small amounts of cytokine molecules intracellularly. Graphene quantum dots modified with cytokine aptamers (Ap-GQDs) and epitope modified GQDs (Ep-GQDs) were prepared; both are normally fluorescent at sufficient dilution. However, the fluorescence of the conjugates of Ap-GQDs and Ep-GQDs is quenched due to aggregation between Ap-GQDs and Ep-GQDs. After incubation of the cytokine-secreting cells with the conjugates of Ap-GQDs and Ep-GQDs, the cytokines secreted in cells compete for binding with the epitope which is then displaced. The ensuing binding of cytokines with the aptamers results in the disaggregation of Ap-GQDs and Ep-GQDs, and the recovery of fluorescence. The conjugates of Ap-GQDs and Ep-GQDs were used as nanosensors for monitoring intracellular cytokine IFN-γ secretion with very high sensitivity (2 pg mL-1). The disaggregation based sensing strategy in this nanosensor design is simple and universal; similar nanosensors can be used for the detection of a broad spectrum of cell-secreted molecules. Such nanosensors will serve as potential biomaterials for in vivo devices to monitor a variety of biological phenomena, in particular to understand cytokine secretion pathways in live cells.


Assuntos
Aptâmeros de Nucleotídeos/química , Citocinas/análise , Grafite , Pontos Quânticos , Animais , Linhagem Celular Tumoral , Fluorescência , Humanos , Interferon gama/análise , Leucócitos Mononucleares , Camundongos , Espectrometria de Fluorescência
17.
Biotechnol Biofuels ; 10: 29, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28184244

RESUMO

BACKGROUND: Solid-binding peptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. RESULTS: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabilities when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation displayed greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates. CONCLUSIONS: The current work introduced the construction of functional biocatalytic modules for the hydrolysis of simple and complex polysaccharides. This technology exploited a silica-binding SBP to mediate effectively the rapid and simple immobilisation of thermostable enzymes onto readily-available and inexpensive silica-based matrices. A conceptual application of biocatalytic modules consisting of single or multiple enzymes was validated by hydrolysing various hemicellulosic polysaccharides.

18.
Acta Biomater ; 51: 461-470, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28063989

RESUMO

The fluorescent protein KillerRed, a new type of biological photosensitizer, is considered as a promising substitute for current synthetic photosensitizes used in photodynamic therapy (PDT). However, broad application of this photosensitiser in treating deep-seated lesions is challenging due to the limited tissue penetration of the excitation light with the wavelength falling in the visible spectral range. To overcome this challenge, we employ upconversion nanoparticles (UCNPs) that are able to convert deep-penetrating near infrared (NIR) light to green light to excite KillerRed locally, followed by the generation of reactive oxygen species (ROS) to kill tumour cells under centimetre-thick tissue. The photosensitizing bio-nanohybrids, KillerRed-UCNPs, are fabricated through covalent conjugation of KillerRed and UCNPs. The resulting KillerRed-UCNPs exhibit excellent colloidal stability in biological buffers and low cytotoxicity in the dark. Cross-comparison between the conventional KillerRed and UCNP-mediated KillerRed PDT demonstrated superiority of KillerRed-UCNPs photosensitizing by NIR irradiation, manifested by the fact that ∼70% PDT efficacy was achieved at 1-cm tissue depth, whereas that of the conventional KillerRed dropped to ∼7%. STATEMENT OF SIGNIFICANCE: KillerRed is a protein photosensitizer that holds promise as an alternative for the existing hydrophobic photosensitizers that are widely used in clinical photodynamic therapy (PDT). However, applications of KillerRed to deep-seated tumours are limited by the insufficient penetration depth of the excitation light in highly scattering and absorbing biological tissues. Herein, we reported the deployment of upconversion nanoparticles (UCNPs) to enhance the treatment depth of KillerRed by converting the deep-penetrating near-infrared (NIR) light to upconversion photoluminescence and activating the PDT effect of KillerRed under deep tissues. This work demonstrated clear potential of UCNPs as the NIR-to-visible light converter to overcome the light penetration limit that has plagued PDT application for many years.


Assuntos
Proteínas de Fluorescência Verde/uso terapêutico , Nanopartículas/química , Fotoquimioterapia , Linhagem Celular Tumoral , Sobrevivência Celular , Endocitose , Transferência de Energia , Humanos , Microscopia Confocal , Nanopartículas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho
19.
Sci Rep ; 6: 27564, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27282464

RESUMO

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.


Assuntos
Quelantes/química , Elementos da Série dos Lantanídeos/química , Luminescência , Proteínas Recombinantes de Fusão/química , Quelantes/farmacologia , Cryptosporidium/isolamento & purificação , Cryptosporidium/patogenicidade , Európio/química , Giardia/isolamento & purificação , Giardia/patogenicidade , Elementos da Série dos Lantanídeos/farmacologia , Ligantes , Medições Luminescentes/métodos , Proteínas Recombinantes de Fusão/farmacologia
20.
ACS Appl Mater Interfaces ; 8(19): 11945-53, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27119593

RESUMO

The treatment depth of existing photodynamic therapy (PDT) is limited because of the absorption of visible excitation light in biological tissue. It can be augmented by means of upconversion nanoparticles (UCNPs) transforming deep-penetrating near-infrared (NIR) light to visible light, exciting PDT drugs. We report here a facile strategy to assemble such PDT nanocomposites functionalized for cancer targeting, based on coating of the UCNPs with a silica layer encapsulating the Rose Bengal photosensitizer and bioconjugation to antibodies through a bifunctional fusion protein consisting of a solid-binding peptide linker genetically fused to Streptococcus Protein G'. The fusion protein (Linker-Protein G) mediates the functionalization of silica-coated UCNPs with cancer cell antibodies, allowing for specific target recognition and delivery. The resulting nanocomposites were shown to target cancer cells specifically, generate intracellular reactive oxygen species under 980 nm excitation, and induce NIR-triggered phototoxicity to suppress cancer cell growth in vitro.


Assuntos
Anticorpos Antineoplásicos , Nanocompostos , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Rosa Bengala , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Nanocompostos/química , Nanocompostos/uso terapêutico , Neoplasias/metabolismo , Neoplasias/patologia , Rosa Bengala/química , Rosa Bengala/farmacologia
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