RESUMEN
We introduce a pH-sensitive amide bond, inspired by citraconic anhydride, for the reversible conjugation of polymers to the lysine residues of proteins and antibodies. The pH sensitivity arises from a conformation lock at the end of the polymer, which we introduce by means of a Diels-Alder reaction, that positions a carboxylic acid close to the amide after conjugation occurs. The amide is stable over weeks at pH 7.4 but sensitive to hydrolysis at pH 5.5 and below, returning the amine to its original state. The pH sensitivity can be tuned by positioning secondary amide groups nearby. We use this approach to PEGylate an antibody to human serum albumin at high dilution and demonstrate successful recovery of the activity after hydrolysis at pH 5.5. These results offer a convenient and traceless approach to protein and antibody functionalization.
Asunto(s)
Anhídridos Citracónicos , Polímeros , Humanos , Anhídridos Citracónicos/química , Concentración de Iones de Hidrógeno , Fenómenos Químicos , Anticuerpos , AmidasRESUMEN
The efficacy of cancer chemotherapy can be generally restrained by the multiple drug resistance (MDR) of tumors, which is typically attributed to the upregulation of ATP-binding cassette (ABC) transporter proteins, such as P-glycoprotein (P-gp). There is an urgent need to present innovative strategies to reverse MDR and enhance the therapeutic efficacy of chemotherapeutic agents in biomedical applications. Here, we report a novel nanosystem of charge-reversal-functionalized PLGA nanobubbles (denoted as Dox-NBs/PPP/P-gp shRNA) for the co-delivery of Dox and P-gp shRNA for the reversal of drug resistance and for ultrasonic-imaging-guided tumor therapy. 1H NMR, FT-IR, SEM, DLS, and UV-vis spectroscopy characterizations were conducted to determine the structure, morphology, and composition of the as-prepared nanobubbles. Dox-NBs/PPP/P-gp shRNA exhibited an average diameter of about 300 nm with good dispersity, biocompatibility, and pH-responsive release properties through the charge-reversal process. The in vitro experiments showed that Dox-NBs/PPP/P-gp shRNA nanobubbles could co-deliver Dox and P-gp shRNA into tumor cells and could effectively suppress P-gp expression, leading to enhanced overall therapeutic effects against MCF-7/MDR cells by restraining the drug efflux. An in vivo antitumor assay revealed an approximately 65% inhibition rate of Dox-NBs/PPP/P-gp shRNA against MCF-7/ADR tumors in tumor-bearing nude mice. Both the in vitro and in vivo toxicity results indicated the Dox-NBs/PPP/P-gp shRNA are highly biocompatible with reducing side-effects and have negligible systemic toxicity in the in vivo therapy of resistant cancers by combining with a chemotherapeutic agent and P-gp knockdown. Furthermore, the in vivo imaging data substantiated that the functionalized nanobubbles could be used as an efficient contrast agent for the ultrasonic imaging of solid tumors. This works highlights the great potential of Dox-NBs/PPP/P-gp shRNA nanobubbles for enhanced imaging-guided combination therapy for overcoming MDR.
Asunto(s)
Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Nanoestructuras/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Anhídridos Citracónicos/administración & dosificación , Anhídridos Citracónicos/química , Terapia Combinada , Doxorrubicina/química , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Nanoestructuras/química , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Polímeros/administración & dosificación , Polímeros/química , ARN Interferente Pequeño/química , Nanomedicina Teranóstica , Carga Tumoral/efectos de los fármacos , UltrasonografíaRESUMEN
BACKGROUND: Anticancer drug-delivery systems (DDSs) capable of responding to the physiological stimuli and efficiently releasing drugs inside tumor cells are highly desirable for effective cancer therapy. Herein, pH-responsive, charge-reversal poly(allylamine hydrochlorid)-citraconic anhydride (PAH-cit) functionalized boron nitride nanospheres (BNNS) were fabricated and used as a carrier for the delivery and controlled release of doxorubicin (DOX) into cancer cells. METHODS: BNNS was synthesized through a chemical vapor deposition method and then functionalized with synthesized charge-reversal PAH-cit polymer. DOX@PAH-cit-BNNS complexes were prepared via step-by-step electrostatic interactions and were fully characterized. The cellular uptake of DOX@PAH-cit-BNNS complexes and DOX release inside cancer cells were visualized by confocal laser scanning microscopy. The in vitro anticancer activity of DOX@ PAH-cit-BNNS was examined using CCK-8 and live/dead viability/cytotoxicity assay. RESULTS: The PAH-cit-BNNS complexes were nontoxic to normal and cancer cells up to a concentration of 100 µg/mL. DOX was loaded on PAH-cit-BNNS complexes with high efficiency. In a neutral environment, the DOX@PAH-cit-BNNS was stable, whereas the loaded DOX was effectively released from these complexes at low pH condition due to amide hydrolysis of PAH-cit. Enhanced cellular uptake of DOX@PAH-cit-BNNS complexes and DOX release in the nucleus of cancer cells were revealed by confocal microscopy. Additionally, the effective delivery and release of DOX into the nucleus of cancer cells led to high therapeutic efficiency. CONCLUSION: Our findings indicated that the newly developed PAH-cit-BNNS complexes are promising as an efficient pH-responsive DDS for cancer therapy.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Nanosferas/administración & dosificación , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacocinética , Compuestos de Boro/química , Anhídridos Citracónicos/química , Doxorrubicina/química , Doxorrubicina/farmacocinética , Liberación de Fármacos , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Células MCF-7 , Nanosferas/química , Poliaminas/química , Polímeros/química , Electricidad EstáticaRESUMEN
The design of highly efficient drug carriers, and the development of appropriate techniques to monitor their mechanism of action and therapeutic effect, are both critical for improving chemotherapy. Herein, a polymeric nanoparticle, PAH-Cit/DOX (poly(allylamine)-citraconic anhydride/doxorubicin), was synthesized and used as a nanodrug system for the efficient delivery and pH-responsive release of doxorubicin (DOX) into cancer cells. The PAH-Cit/DOX nanoparticles were stable at physiological pH but effectively released DOX under weakly acidic conditions. The release efficiency was 90.6% after 60 h of dialysis in phosphate-buffered saline at pH 5.5. Confocal images showed the rapid movement of the drug from the cytoplasm to the nucleus, indicating the effective drug release MCF-7 cells. Notably, the combination of fluorescence lifetime imaging microscopy (FLIM) and phasor analysis (phasor-FLIM) provides an approach to monitor the dynamic change of DOX fluorescence lifetime in intercellular environments. Phasor-differentiated lifetime pixel intensity in FLIM images was quantified and used to evaluate the DOX release from nanocarriers, making it possible to detect the dynamics of intracellular release and transport of DOX.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Anhídridos Citracónicos/química , Doxorrubicina/farmacología , Portadores de Fármacos/química , Nanopartículas/química , Poliaminas/química , Polímeros/química , Polipropilenos/química , Antibióticos Antineoplásicos/química , Doxorrubicina/química , Portadores de Fármacos/síntesis química , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Microscopía Confocal , Microscopía Fluorescente , Nanopartículas/toxicidad , Polímeros/síntesis química , Polímeros/toxicidad , Polipropilenos/síntesis química , Polipropilenos/toxicidadRESUMEN
Increasing attention has been paid to nasal delivery. Subunit vaccines based on antigenic proteins or polypeptides offer good safety. However, lack of delivery efficiency, particularly for nasal immunization, is a big issue. Here we designed a highly tunable polymeric hybrid micelle (PHM) system offering good vaccine efficacy after nasal administration. PHMs are formulated from two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG), the ratio of which determines PHM physicochemical properties. Citraconic anhydride-modified ovalbumin (Cit-OVA), as model antigen, was incorporated into PHMs via electrostatic interaction, giving antigen-loaded micelles of around 150nm in size. Their surface characteristics which are found closely related to their in vivo kinetics can be modulated by adjusting the mass ratio of PCL-PEG and PCL-PEI. PHM/Cit-OVA complexes containing PCL-PEI and PCL-PEG in a 1:1 mass ratio induced strong immune responses in nasal mucosa and serum in vivo without causing obvious toxicity, and Cit-OVA was efficiently internalized by dendritic cells. These results demonstrate the promise of this multifunctional polymeric delivery system for nasal vaccination.
Asunto(s)
Antígenos/administración & dosificación , Anhídridos Citracónicos/administración & dosificación , Micelas , Ovalbúmina/administración & dosificación , Polímeros/administración & dosificación , Administración Intranasal , Animales , Antígenos/química , Línea Celular , Anhídridos Citracónicos/química , Células Dendríticas/efectos de los fármacos , Perros , Femenino , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Ganglios Linfáticos/citología , Células de Riñón Canino Madin Darby , Ratones Endogámicos BALB C , Membrana Mucosa/inmunología , Ovalbúmina/química , Polímeros/químicaRESUMEN
A boronate ester crosslinked zwitterionic nanogel (NGCA) with ATP/pH-sensitivity has been developed with an inverse nanoprecipitation technique to achieve a two-stage charge conversion that responds to tumor extracellular conditions (pH 6.5-6.8) and an intracellular acidic environment (pH 5-6). Cationic cytochrome C (CC), a therapeutic protein, has been encapsulated into NGCA through inverse nanoprecipitation via electrostatic interactions to form protein-loaded nanogel (NGCA-CC). By adjusting the ratio of the amino and carboxyl groups in the nanogels, negatively charged nanogels that are safer under physiological conditions (pH 7.4) can convert their surface charge to positive at tumor extracellular pH, which enhance their cellular uptake efficiency. The citraconic amide formed from citraconic anhydride and amine can be cleaved in the intracellular acidic organelles to expose more amino groups and facilitate endosomal escape. The release of CC is accelerated in the presence of 5 mM ATP or under acidic conditions. Confocal laser scanning microscopy (CLSM) and flow cytometry have shown that NGCA-CC's cell uptake is higher at pH 6.5 than at pH 7.4. MTT and real-time cell analysis (RTCA) have illustrated that there is more toxicity at pH 6.5 than at pH 7.4. The apoptosis process induced by CC was determined by flow cytometry.
Asunto(s)
Ácidos Borónicos/química , Anhídridos Citracónicos/química , Citocromos c/química , Citocromos c/uso terapéutico , Nanopartículas/química , Polietilenglicoles/química , Polietileneimina/química , Apoptosis/efectos de los fármacos , Citocromos c/metabolismo , Portadores de Fármacos/química , Citometría de Flujo/métodos , Humanos , Concentración de Iones de Hidrógeno , Nanogeles , NanotecnologíaRESUMEN
Polyampholyte-based films can be efficiently self-assembled onto a surface in a one-pot manner. By using a gradient of protons, morphogens, generated at an electrode surface, a charge-shifting polyelectrolyte present in solution can be transformed into a polyampholyte, leading to the continuous buildup of a film based on polyelectrolyte complexation.
Asunto(s)
Anhídridos Citracónicos/química , Electrólitos/química , Poliaminas/química , Polímeros/química , Estructura Molecular , Polielectrolitos , Electricidad Estática , Propiedades de SuperficieRESUMEN
Multifunctional nanocomplexes (NCs) consisting of urocanic acid-modified galactosylated trimethyl chitosan (UA-GT) conjugates as polymeric vectors, poly(allylamine hydrochloride)-citraconic anhydride (PAH-Cit) as charge-reversible crosslinkers, and vascular endothelial growth factor (VEGF) siRNA as therapeutic genes, were rationally designed to simultaneously overcome the extracellular, cellular, and intracellular barriers for siRNA delivery. The strong physical stability of UA-GT/PAH-Cit/siRNA NCs (UA-GT NCs) at pH 7.4 and 6.5 endowed protection from massive dilution, competitive ions, and ubiquitous nucleases in the blood and tumorous microenvironment. Their internalization into hepato-carcinoma cells was facilitated through the recognition of galactose receptors, followed by effective escape from endosomes/lysosomes owing to the strong buffering capacity of imidazole residues. At the meantime, the endosomal/lysosomal acidity triggered the charge reversal of PAH-Cit in UA-GT NCs, thus evoking their structural disassembly and subsequently accelerated release of siRNA in the cytosol. As a result, robust in vivo performance in terms of both gene silencing and tumor inhibition was achieved by UA-GT NCs at a low siRNA dose. Moreover, neither histological nor hematological toxicity was detected following repeated intravenous administration. Therefore, UA-GT NCs potentially served as an efficient and safe candidate in the treatment of hepatocellular carcinoma through knocking down the overall barriers for siRNA delivery.
Asunto(s)
Antineoplásicos/farmacología , Técnicas de Transferencia de Gen , Nanopartículas/química , ARN Interferente Pequeño/metabolismo , Animales , Tampones (Química) , Línea Celular Tumoral , Quitosano/síntesis química , Quitosano/química , Anhídridos Citracónicos/síntesis química , Anhídridos Citracónicos/química , Endocitosis , Endosomas/metabolismo , Femenino , Galactosa/química , Silenciador del Gen/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/toxicidad , Tamaño de la Partícula , Poliaminas/síntesis química , Poliaminas/química , Espectroscopía de Protones por Resonancia Magnética , Electricidad Estática , Ácido Urocánico/síntesis química , Ácido Urocánico/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Therapeutic platforms such as chemotherapy that respond to physical and biological stimuli are highly desirable for effective cancer therapy. In this study, pH-responsive charge-reversal, polymer-coated mesoporous silica nanoparticles [PAH-cit/APTES-MSNs; PAH-cit refers to poly(allylamine)-citraconic anhydride; APTES refers to (3-aminopropyl)triethoxysilane] were synthesized for application as drug-delivery systems for the treatment of malignant cells. Confocal laser scanning microscopy (CLSM) revealed that the PAH-cit/APTES-MSNs nanocomposite effectively delivered and released doxorubicin hydrochloride to the nucleus of HeLa (human cervical carcinoma) cells. Additionally, the real-time dynamic drug-release process was monitored by CLSM. The current pH-controlled-smart-release platform holds promise in drug-delivery and cancer therapy-related applications.
Asunto(s)
Materiales Biocompatibles Revestidos/química , Preparaciones de Acción Retardada/farmacología , Liberación de Fármacos , Espacio Intracelular/química , Nanopartículas/química , Polímeros/química , Dióxido de Silicio/química , Muerte Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Anhídridos Citracónicos/química , Doxorrubicina/farmacología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Microscopía Confocal , Nanopartículas/ultraestructura , Poliaminas/química , Porosidad , Propilaminas , Silanos/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Our previous work showed that a charge-reversal layer-by-layer nanosystem, PEI/PAH-Cit/AuNP-CS, effectively facilitates cellular uptake of siRNA and enhances the silencing efficacy of MDR1 siRNA. Here, the plasmid loading capacity of this vehicle was examined using EGFP-N1, and the plasmid release profile was determined in response to pH changes. The cytotoxicity of the EGFP-N1/PEI/PAH-Cit/AuNP-CS complex against HeLa and 293T cells was almost negligible. PEI/PAH-Cit/AuNP-CS efficaciously delivered the plasmids EGFP-N1 (encoding green fluorescent protein) and pGL3.0 (encoding luciferase) into 293T and HeLa cells, thus verifying the universality of PEI/PAH-Cit/AuNP-CS as a gene carrier. The results of an inverted fluorescence microscopy, flow cytometry (FCM) and western blotting methods demonstrated that PC-3 prostate cancer cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS expressed higher levels of GFP than cells treated with EGFP-p53/PEI. Furthermore, PC-3 cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS showed reduced cellular viability and increased nuclear fragmentation, consistent with elevated p53 expression. Propidium iodide (PI) flow cytometric assays were conducted to demonstrate that EGFP-p53/PEI/PAH-Cit/AuNP-CS elevated the level of apoptosis in PC-3 cells. Western blotting and caspase activation studies revealed that EGFP-p53/PEI/PAH-Cit/AuNP-CS complexes may induce PC-3 apoptosis via the mitochondria-mediated signaling pathway by up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3.
Asunto(s)
Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Proliferación Celular , Supervivencia Celular , Quitosano/química , Anhídridos Citracónicos/química , Oro/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Masculino , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Microscopía Fluorescente , Plásmidos/metabolismo , Poliaminas/química , Polietileneimina/química , Neoplasias de la Próstata/patología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Tannerella forsythensis (Bacteroides forsythus), an anaerobic Gram-negative species of bacteria that plays a role in the progression of periodontal disease, has a unique bacterial protein profile. It is characterized by two unique protein bands with molecular weights of more than 200 kDa. It also is known to have a typical surface layer (S-layer) consisting of regularly arrayed subunits outside the outer membrane. We examined the relationship between high molecular weight proteins and the S-layer using electron microscopic immunolabeling with chemical fixation and an antigen retrieval procedure consisting of heating in a microwave oven or autoclave with citraconic anhydride. Immunogold particles were localized clearly at the outermost cell surface. We also used energy-filtering transmission electron microscopy (EFTEM) to visualize 3, 3'-diaminobenzidine tetrahydrochloride (DAB) reaction products after microwave antigen retrieval with 1% citraconic anhydride. The three-window method for electron spectroscopic images (ESI) of nitrogen by the EFTEM reflected the presence of moieties demonstrated by the DAB reaction with horseradish peroxidase (HRP)-conjugated secondary antibodies instead of immunogold particles. The mapping patterns of net nitrogen were restricted to the outermost cell surface.
Asunto(s)
Antígenos Bacterianos/análisis , Bacteroides/inmunología , Bacteroides/ultraestructura , Anhídridos Citracónicos/química , Calefacción/instrumentación , Inmunohistoquímica/métodos , Glicoproteínas de Membrana/análisis , Energía Filtrada en la Transmisión por Microscopía Electrónica/métodos , Microondas , 3,3'-Diaminobencidina/química , Proteínas Bacterianas/análisis , Bacteroides/química , Peroxidasa de Rábano Silvestre/químicaRESUMEN
Intracellular delivery of active proteins presents an interesting approach in research and therapy. We created a protein transduction shuttle based on a new traceless click linker that combines the advantages of click reactions with implementation of reversible pH-sensitive bonds. The azidomethyl-methylmaleic anhydride (AzMMMan) linker was found compatible with different click chemistries, demonstrated in bioreversible protein modification with dyes, polyethylene glycol, or a transduction carrier. Linkages were stable at physiological pH but reversible at the mild acidic pH of endosomes or lysosomes. We show that pH-reversible attachment of a defined endosome-destabilizing three-arm oligo(ethane amino)amide carrier generates an effective shuttle for protein delivery. The cargo protein nlsEGFP, when coupled via the traceless AzMMMan linker, experiences efficient cellular uptake and endosomal escape into the cytosol, followed by import into the nucleus. In contrast, irreversible linkage to the same shuttle hampers nuclear delivery of nlsEGFP which after uptake remains trapped in the cytosol. Successful intracellular delivery of bioactive ß-galactosidase as a model enzyme was also demonstrated using the pH-controlled shuttle system.
Asunto(s)
Anhídridos Citracónicos/química , Portadores de Fármacos/química , Proteínas Fluorescentes Verdes/administración & dosificación , beta-Galactosidasa/administración & dosificación , Azidas/química , Azidas/metabolismo , Permeabilidad de la Membrana Celular , Anhídridos Citracónicos/metabolismo , Química Clic , Citosol/metabolismo , Portadores de Fármacos/metabolismo , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/farmacocinética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , beta-Galactosidasa/química , beta-Galactosidasa/farmacocinéticaAsunto(s)
Dendrímeros/química , Portadores de Fármacos , Colorantes Fluorescentes/metabolismo , Neoplasias Hepáticas/metabolismo , Oxazinas/metabolismo , Animales , Transporte Biológico , Química Farmacéutica , Ácido Cólico/química , Anhídridos Citracónicos/química , Preparaciones de Acción Retardada , Composición de Medicamentos , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos BALB C , Micelas , Microscopía Confocal , Oxazinas/administración & dosificación , Oxazinas/química , Espectroscopía Infrarroja Corta , Tecnología Farmacéutica/métodos , Factores de Tiempo , Distribución Tisular , Imagen de Cuerpo EnteroRESUMEN
We report a smart mesoporous silica nanoparticle (MSN) with a pore surface designed to undergo charge conversion in intracellular endosomal condition. The surface of mesopores in the silica nanoparticles was engineered to have pH-hydrolyzable citraconic amide. Solid-state nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR) spectroscopy, and Brunauer-Emmett-Teller (BET) analyses confirmed the successful modification of the pore surfaces. MSNs (MSN-Cit) with citraconic amide functionality on the pore surfaces exhibited a negative zeta potential (-10 mV) at pH 7.4 because of the presence of carboxylate end groups. At cellular endosomal pH (approximately 5.0), MSN-Cit have a positive zeta potential (16 mV) indicating the dramatic charge conversion from negative to positive by hydrolysis of surface citraconic amide. Cytochrome c (Cyt c) of positive charges could be incorporated into the pores of MSN-Cit by electrostatic interactions. The release of Cyt c can be controlled by adjusting the pH of the release media. At pH 7.4, the Cyt c release was retarded, whereas, at pH 5.0, MSN-Cit facilitated the release of Cyt c. The released Cyt c maintained the enzymatic activity of native Cyt c. Hemolytic activity of MSN-Cit over red blood cells (RBCs) was more pronounced at pH 5.0 than at pH 7.0, indicating the capability of intracellular endosomal escape of MSN carriers. Confocal laser scanning microscopy (CLSM) studies showed that MSN-Cit effectively released Cyt c in endosomal compartments after uptake by cancer cells. The MSN developed in this work may serve as efficient intracellular carriers of many cell-impermeable therapeutic proteins.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Proteínas/administración & dosificación , Proteínas/química , Dióxido de Silicio/química , Adsorción , Supervivencia Celular , Anhídridos Citracónicos/química , Anhídridos Citracónicos/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Células HeLa , Hemólisis , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Modelos Químicos , Nitrógeno , Porosidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: The introduction of heat-induced antigen retrieval has been a major milestone in diagnostic immunohistochemistry, enabling the application of many antibodies to fixed paraffin-embedded tissue sections. A number of important variables affect the preservation of tissue antigens, among which are analytical variables including the antigen retrieval methodology. Temperature of retrieval, duration of heating, source of heat, pH and nature of retrieval solution are among more important variables pivotal to results. Citrate buffer at pH 6.0 has become widely embraced as the universal fixative but some antibodies remain capricious and yield poor staining results. METHODS: This study examines the recent suggestion that citraconic anhydride may be a suitable universal retrieval reagent. Immunostaining of 65 commonly employed antibodies following microwave antigen retrieval in 0.05% citraconic anhydride for 10 minutes at 98 degrees C was compared with consecutive tissues sections subjected to antigen retrieval in citrate buffer at pH 6.0 at the same duration and temperature. RESULTS: Thirty-five of the 65 antibodies examined yielded more intense staining following antigen retrieval in citraconic anhydride, including some capricious antibodies such as MyoD1, myogenin, perforin, TIA-1, Tdt, RET and MiTF, confirming the efficacy of this retrieval solution. CONCLUSION: It is recommended that consideration be given to 0.05% citraconic anhydride as an antigen retrieval solution, particularly for antibodies that fail to work or stain weakly in fixed paraffin-embedded tissue sections.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Antígenos/química , Anhídridos Citracónicos/química , Fijadores/química , Técnicas para Inmunoenzimas/métodos , Fijación del Tejido/métodos , Biomarcadores/química , HumanosRESUMEN
Insulin is a polypeptide hormone which is produced by the beta-cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des-threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des-threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2-3 at 25 degrees C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des-threonine insulin and hence increased the production yield of active insulin.
Asunto(s)
Carboxipeptidasa B/química , Anhídridos Citracónicos/química , Proinsulina/química , Procesamiento Proteico-Postraduccional , Tripsina/química , Secuencia de Aminoácidos , Carboxipeptidasa B/metabolismo , Humanos , Datos de Secuencia Molecular , Tripsina/metabolismoRESUMEN
The phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes forms aggregates with anionic lipids leading to low activity. The specific activity of the enzyme can be enhanced by dilution of the protein or by addition of both zwitterionic and neutral amphiphiles (e.g., diheptanoylphosphatidylcholine or Triton X-100) or 0.1-0.2 M inorganic salts. Activation by amphiphiles occurs with both micellar (phosphatidylinositol dispersed in detergents) and monomeric [dibutroylphosphatidylinositol (diC(4)PI)] phosphotransferase substrates and inositol 1,2-(cyclic)-phosphate (cIP), the phosphodiesterase substrate. The presence of zwitterionic and neutral amphiphiles (to which the protein binds weakly) dilutes the surface concentration of the interfacial anionic substrate and thereby reduces the level of enzyme-phospholipid particle aggregation. Zwitterionic amphiphiles also can bind directly to the protein and enhance catalysis since they enhance both diC(4)PI and cIP hydrolysis. In contrast to activation by amphiphiles, the rate enhancement by salt occurs for only the phosphotransferase step of the reaction. Added salt has a synergistic effect with zwitterionic phospholipids, leading to high specific activities for PI cleavage with only moderate dilution of the anionic substrate in the interface. This kinetic activation correlates with weakening of strong PI-PLC hydrophobic interactions with the interface as monitored by a decrease in the maximum monolayer surface pressure for insertion of the protein. Several point mutations of surface hydrophobic residues (W49A, L51A, L235A, and F237W) can dramatically alter the unusual kinetics of this secreted enzyme. The high affinity of PI-PLC for anionic phospholipids along with a strong hydrophobic interaction, which gives rise to the unusual kinetic behavior, is considered in terms of how it might contribute to the role of this phospholipase in L. monocytogenes infectivity.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Listeria monocytogenes/enzimología , Fosfoinositido Fosfolipasa C/química , Fosfoinositido Fosfolipasa C/metabolismo , Animales , Proteínas Bacterianas/genética , Anhídridos Citracónicos/química , Detergentes/química , Activación Enzimática , Listeria monocytogenes/patogenicidad , Modelos Moleculares , Fosfoinositido Fosfolipasa C/genética , Conformación ProteicaRESUMEN
Special delivery! Polyionic complex (PIC) micelles that contain the charge-conversional moieties citaconic amide or cis-aconitic amide were developed for cytoplasmic protein delivery. The increase of the charge density on the protein cargo helped the stability of the PIC micelles without cross-linking, and the charge-conversion in endosomes induced the dissociation of the PIC micelles to result in efficient endosomal release (see picture).
Asunto(s)
Anhídridos Citracónicos/química , Citocromos c/administración & dosificación , Citocromos c/química , Portadores de Fármacos/química , Micelas , Amidas/química , Animales , Ácido Aspártico/química , Línea Celular Tumoral , Citocromos c/farmacocinética , Citoplasma/metabolismo , Corazón , Caballos , Humanos , Iones/química , Miocardio/enzimología , Polietilenglicoles/químicaRESUMEN
BACKGROUND: Enzymes show relative instability in solvents or at elevated temperature and lower activity in organic solvent than in water. These limit the industrial applications of enzymes. RESULTS: In order to improve the activity and stability of chloroperoxidase, chloroperoxidase was modified by citraconic anhydride, maleic anhydride or phthalic anhydride. The catalytic activities, thermostabilities and organic solvent tolerances of native and modified enzymes were compared. In aqueous buffer, modified chloroperoxidases showed similar Km values and greater catalytic efficiencies kcat/Km for both sulfoxidation and oxidation of phenol compared to native chloroperoxidase. Of these modified chloroperoxidases, citraconic anhydride-modified chloroperoxidase showed the greatest catalytic efficiency in aqueous buffer. These modifications of chloroperoxidase increased their catalytic efficiencies for sulfoxidation by 12%~26% and catalytic efficiencies for phenol oxidation by 7%~53% in aqueous buffer. However, in organic solvent (DMF), modified chloroperoxidases had lower Km values and higher catalytic efficiencies kcat/Km than native chloroperoxidase. These modifications also improved their thermostabilities by 1~2-fold and solvent tolerances of DMF. CD studies show that these modifications did not change the secondary structure of chloroperoxidase. Fluorescence spectra proved that these modifications changed the environment of tryptophan. CONCLUSION: Chemical modification of epsilon-amino groups of lysine residues of chloroperoxidase using citraconic anhydride, maleic anhydride or phthalic anhydride is a simple and powerful method to enhance catalytic properties of enzyme. The improvements of the activity and stability of chloroperoxidase are related to side chain reorientations of aromatics upon both modifications.