RESUMO
With a growing number of intracellular drug targets and the high efficacy of protein therapeutics, the targeted delivery of active proteins with negligible toxicity is a challenging issue in the field of precision medicine. Herein, a programed assembly of nucleoprotein nanoparticles (NNPs) using DNA and zinc fingers (ZnFs) for targeted protein delivery is presented. Two types of ZnFs with different sequence specificities are genetically fused to a targeting moiety and a protein cargo, respectively. Double-stranded DNA with multiple ZnF-binding sequences is grafted onto inorganic nanoparticles, followed by conjugation with the ZnF-fused proteins, generating the assembly of NNPs with a uniform size distribution and high stability. The approach enables controlled loading of a protein cargo on the NNPs, offering a high cytosolic delivery efficiency and target specificity. The utility and potential of the assembly as a versatile protein delivery vehicle is demonstrated based on their remarkable antitumor activity and target specificity with negligible toxicity in a xenograft mice model.
Assuntos
DNA/química , Nanopartículas/química , Nucleoproteínas/química , Proteínas/química , Animais , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Ligação Proteica , Dedos de ZincoRESUMO
Antibody-mimetic proteins are intensively being developed for biomedical applications including tumor imaging and therapy. Among them, repebody is a new class of protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His6)-tag bearing repebody (rEgH9) was labeled with [99mTc]-tricarbonyl, and biodistribution was performed following intravenous (I.V.) or intraperitoneal (I.P.) injection. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. Biodistribution data indicates radiotracer has a rapid clearance from blood and excreted through the kidneys for intravenous (I.V.) injection, but comparatively slow clearance for an intraperitoneal (I.P.) injection. SPECT-CT images were found to be in agreement with biodistribution data, high activity was found inside kidneys. The observed result for rapid blood clearance and renal excretion of repebody (rEgH9) provide useful information for the further development of therapeutic strategy.
Assuntos
Proteínas/química , Compostos Radiofarmacêuticos/química , Tecnécio/química , Administração Intravenosa , Animais , Anticorpos/química , Anticorpos/metabolismo , Infusões Parenterais , Marcação por Isótopo , Rim/diagnóstico por imagem , Rim/metabolismo , Leucina/química , Leucina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (72±8%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules.
Assuntos
Alcinos/química , Azidas/química , Química Click , Compostos Radiofarmacêuticos/química , Animais , Cobre/química , Radioisótopos do Iodo/química , Marcação por Isótopo , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/química , Compostos Radiofarmacêuticos/síntese química , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Herein we report an efficient method for iodine radioisotope labeling of cyclooctyne-containing molecules using copper-free click reaction. For this study, radioiodination using the tin precursor 2 was carried out at room temperature to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (85%) and excellent radiochemical purity. Dibenzocyclooctyne (DBCO) containing cRGD peptide and gold nanoparticle were labeled with [(125)I]1 at 37°C for 30min to give triazoles with good radiochemical yields (67-95%). We next carried out tissue biodistribution study of [(125)I]1 in normal ICR mice to investigate the level of organ accumulation which needs to be considered for pre-targeted in vivo imaging. Large amount of [(125)I]1 distributed rapidly in liver and kidney from bloodstream and underwent rapid renal and hepatobiliary clearance. Moreover [(125)I]1 was found to be highly stable (>92%) in mouse serum for 24h. Therefore [(125)I]1 could be used as a potentially useful radiotracer for pre-targeted imaging. Those results clearly indicated that the present radiolabeling method using copper free click reaction would be quite useful for both in vitro and in vivo labeling of DBCO group containing molecules with iodine radioisotopes.
Assuntos
Azidas/química , Ciclo-Octanos/química , Ouro/química , Marcação por Isótopo/métodos , Compostos Radiofarmacêuticos/síntese química , Animais , Química Click , Radioisótopos do Iodo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/química , Compostos Radiofarmacêuticos/sangue , Compostos Radiofarmacêuticos/farmacocinéticaRESUMO
In this report, the novel and site-specific radioiodination of biomolecules by using aryl diamine and alkyl aldehyde condensation reaction in the presence of a Cu2+ catalyst under ambient conditions was reported. 125I-labeled alkyl aldehyde was synthesized using a tin precursor with a high radiochemical yield (72 ± 6%, n = 5) and radiochemical purity (>99%). The utility of the radioiodinated precursor was demonstrated through aryl diamine-installed c[RGDfK(C)] peptide and human serum albumin (HSA). Radioiodinated c[RGDfK(C)] peptide and HSA protein were synthesized with high radiochemical yields and purity. 125I-HSA protein showed excellent in vivo stability and negligible thyroid uptake as compared with directly radioiodinated HSA by using the tyrosine group. Excellent reaction kinetics and the in vitro and in vivo stabilities of 125I-labeled alkyl aldehyde have suggested the usefulness of the strategy for the radioiodination of bioactive molecules.
RESUMO
Small-sized non-antibody scaffolds have attracted considerable interest as alternatives to immunoglobulin antibodies. However, their short half-life is considered a drawback in the development of therapeutic agents. Here we demonstrate that a homo-dimeric form of a repebody enhances the anti-tumor activity than a monomeric form through prolonged blood circulation. Spytag and spycatcher were genetically fused to the C-terminus of a respective human IL-6-specific repebody, and the resulting two repebody constructs were mixed at an equimolar ratio to produce a homo-dimeric form through interaction between spytag and spycatcher. The homo-dimeric repebody was detected as a single band in the SDS-PAGE analysis with an expected molecular size (78â¯kDa), showing high stability and homogeneity. The dimeric repebody was shown to simultaneously accommodate two hIL-6 molecules, and its binding affinity for hIL-6 was estimated to be comparable to a monomeric repebody. The serum concentration of the dimeric repebody was observed to be about 5.5 times higher than a monomeric repebody, consequently leading to considerably higher tumor suppression effect in human tumor xenograft mice. The present approach can be effectively used for prolonging the blood half-life of small-sized protein binders, resulting in enhanced therapeutic efficacy.
Assuntos
Antineoplásicos/administração & dosagem , Interleucina-6/metabolismo , Peptídeos/química , Proteínas/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gamma irradiation is able to affect various structural modification and an increase of the biological properties of biomaterials. This study was conducted to investigate the anti-allergenic effect of γ-irradiated black ginseng extract (BGE) using in vitro and in vivo experiments. IgEantigen complex-induced degranulation was measured in RBL-2H3 mast cells. In addition, an anti-atopic dermatitis (AD) test was carried out by spreading γ-irradiated BGE on the dorsal skin of 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice. The content of arginylfructose (AF) of gamma-irradiated BGE was higher than that of BGE. In RBL-2H3 mast cells, γ-irradiated BGE treatments significantly reduced the IgE-antigen complex-induced release of ß-hexosaminidase, histamine, intracellular ROS, and Ca2+ influx. A western blot analysis showed that γ-irradiated BGE had an inhibitory activity on the FcεRI-mediated signaling in mast cells. In the DNCB-induced AD model, γ-irradiated BGE significantly alleviated the ADlike skin symptoms and clinical signs. The suppression of AD by γ-irradiated BGE was accompanied by a decrease in the serum level of IgE and IL-4, as well as the number of leukocyte. Gamma-irradiated BGE also suppressed IL-4 and increased IFN-γ in splenocytes. Our data suggests that γ-irradiated BGE may be effective therapeutic agents for the treatment of AD.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dermatite Atópica/prevenção & controle , Raios gama , Mastócitos/efeitos dos fármacos , Panax/química , Extratos Vegetais/química , Animais , Técnicas de Cultura de Células , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/patologia , Dinitroclorobenzeno/toxicidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/efeitos da radiaçãoRESUMO
Luteolin was gamma irradiated at doses of 0, 15, 30, 50, 70, and 100 kGy. We observed that the luteolin peak decreased simultaneously with the appearance of new radiolytic peaks, using high-performance liquid chromatography (HPLC). The highest new radiolytic peak (GLM) of radiolytic product in gamma-irradiated luteolin was observed at a dose of 70 kGy, and the GLM was identified by nuclear magnetic resonance and high-performance-liquid-chromatography-quadrupole-time-of-flight (HPLC-Q-TOF) mass spectrometry. We examined whether 70 kGy gamma-irradiated luteolin has more effective anti-melanogenic effects than intact luteolin. Seventy kilograys of gamma-irradiated luteolin inhibited melanin synthesis and intracellular tyrosinase activity without cytotoxicity, whereas the intact luteolin-treated group did not show anti-melanogenic activity in 3-isobutyl-1-methylxanthine-stimulated B16BL6 melanoma cells. The expression of melanogenic enzymes, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, was decreased by 70 kGy gamma-irradiated luteolin treatment, owing to the suppression of microphthalamia-associated transcription factor and 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein. In addition, gamma-irradiated luteolin decreased the phosphorylation of phosphoinositide 3-kinase (PI3K)/Akt and extracellular regulated kinase (ERK). The anti-melanogenic effects of 70 kGy gamma-irradiated luteolin were attenuated by the treatment of two specific inhibitors (PD98059 and LY294002), and these results indicate that the anti-melanogenic effects were mediated by ERK and PI3K signaling pathways. Therefore, our findings suggest that gamma-irradiated luteolin can be a potential cosmeceutical agent for skin whitening.
Assuntos
1-Metil-3-Isobutilxantina/toxicidade , Luteolina/farmacologia , Melaninas/metabolismo , Melanoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Raios gama , Humanos , Luteolina/efeitos da radiação , Melanoma/induzido quimicamente , Melanoma/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.
Assuntos
ADP Ribose Transferases/química , Antineoplásicos Fitogênicos/administração & dosagem , Toxinas Bacterianas/química , Portadores de Fármacos/química , Exotoxinas/química , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Fatores de Virulência/química , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Domínios Proteicos , Proteínas Inativadoras de Ribossomos Tipo 1/uso terapêutico , Exotoxina A de Pseudomonas aeruginosaRESUMO
The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment.
Assuntos
Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/farmacocinética , Terapia de Alvo Molecular/métodos , Nanopartículas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Engenharia de Proteínas/métodos , Animais , Antineoplásicos/administração & dosagem , Proteínas do Capsídeo/genética , Linhagem Celular Tumoral , Cristalização/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/patologia , Terapia Viral Oncolítica/métodos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Resultado do TratamentoRESUMO
This study was conducted to evaluate the preventive effect of hesperetin against radiation-induced DNA damage and immune dysfunction in murine splenocytes. Isolated splenocytes from BALB/c mice were treated with hesperetin (20, 100, and 500 µM), and then irradiated at a dose of 2 and 4 Gy of γ-irradiation. Exposure to ?-radiation resulted in DNA damage and a reduction of cell viability as well as an elevation of the levels of proinflammatory cytokines, intracellular ROS (reactive oxygen species), and NO (nitric oxide). Hesperetin significantly enhanced the cell viability of the splenocytes compared with the irradiated group. In addition, hesperetin was found to be highly effective in preventing DNA damage as identified by comet and DNA ladder assays. Hesperetin also effectively inhibited proinflammatory cytokines, intracellular ROS, and NO in irradiated splenocytes. In conclusion, hesperetin was shown to be radioprotective against irradiation-induced DNA damage and immune dysfunction in murine splenocytes.
RESUMO
The present study was designed to evaluate the radioprotective effect of hesperidin, a citrus flavanoglycone, against γ-radiation-induced cellular damage in the liver, heart, and kidney of rats. Whole-body γ-radiation exposure (5 Gy) of healthy adult rats resulted in cellular damage and oxidative stress manifested as increased levels of serum marker enzymes, lipid peroxidation, and fibrosis in the tissues, accompanied by depletion of cellular glutathione and abnormal alteration in the levels of lysosomal enzymes. Treatment with hesperidin (50 and 100 mg/kg, p.o.) for 7 days was found to offer significant protection against γ-radiation-induced toxicity in the tissues, which was evident by the improved status of most of the parameters investigated. Further, the histological examination of periodic acid-Schiff-stained tissue sections of animals treated with hesperidin following radiation exposure showed minimal necrotic damage with a recovery pattern in a dose-dependent manner compared with radiation-exposed animals. The results of our study show that administration of hesperidin offers effective protection against γ-radiation-induced cellular damage and oxidative stress in rats.