RESUMO
7-Ethyl-10-hydroxycamptothecin (SN-38) loaded poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F-108) and poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) nanoparticles were successfully prepared by a modified film hydration method and characterized by scanning electric microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). Satisfactory drug loading of 20.73 ± 0.66% and a high encapsulation efficiency of 83.83 ± 1.32% were achieved. The SN-38 nanoparticles (SN-38 NPs) can completely disperse into a phosphate buffered saline (PBS) medium to produce a clear aqueous suspension that remains stable for up to three days. Total drug releases were 67.91% and 91.09% after 24 h in a PBS or fetal bovine serum (FBS) medium. Half maximal inhibitory concentration (IC(50)) tests of SN-38 and SN-38 NPs on A549 lung cells produced results of 200.0 ± 14.9 ng ml(-1) and 80.0 ± 4.6 ng ml(-1), respectively. Similarly, IC(50) tests of SN-38 and SN-38 NPs on MCF-7 breast cells yielded results of 16.0 ± 0.7 ng ml(-1) and 8.0 ± 0.5 ng ml(-1), respectively. These in vitro IC(50) studies show significant (p < 0.01) enhancement of the SN-38 NP drug efficiency in killing cancer cells in comparison to the free drug SN-38 control. All the materials used for this nanoformulation are approved by the US FDA, with the virtue of extremely low toxicity to normal cells.
Assuntos
Camptotecina/análogos & derivados , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Neoplasias Experimentais/tratamento farmacológico , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Camptotecina/administração & dosagem , Camptotecina/química , Sobrevivência Celular/efeitos dos fármacos , Difusão , Humanos , Irinotecano , Micelas , Neoplasias Experimentais/metabolismo , Células Tumorais CultivadasRESUMO
We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-D-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the (18)F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Desoxiglucose/análogos & derivados , Ouro/uso terapêutico , Nanopartículas/uso terapêutico , Adenocarcinoma/patologia , Adenocarcinoma/radioterapia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Mama/efeitos dos fármacos , Mama/patologia , Mama/efeitos da radiação , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Desoxiglucose/química , Desoxiglucose/farmacocinética , Desoxiglucose/uso terapêutico , Feminino , Ouro/química , Ouro/farmacocinética , Camundongos , Nanopartículas/química , Radiossensibilizantes/química , Radiossensibilizantes/farmacocinética , Radiossensibilizantes/uso terapêutico , Compostos de Sulfidrila/químicaRESUMO
The mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell's kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.
Assuntos
Técnicas Biossensoriais/métodos , Proliferação de Células , Células/química , Ensaios de Triagem em Larga Escala/métodos , Hipoxantina Fosforribosiltransferase/genética , Mutação , Animais , Células/citologia , Células/enzimologia , Cricetinae , Hipoxantina Fosforribosiltransferase/metabolismo , CinéticaRESUMO
PURPOSE: MicroRNAs (miRNAs) post-transcriptionally regulate hundreds of gene targets involved in tumorigenesis thereby controlling vital biological processes, including cellular proliferation, differentiation and apoptosis. MiRNA profiling is an emerging tool for the potential early detection of a variety of malignancies. This study was conducyed to assess the feasibility and methodological robustness of quantifying sputum miRNAs, employing quantitative real-time polymerase chain reaction (RT-qPCR) and cluster analysis on an optimized miRNA profile as a novel approach for the early detection of non-small cell lung cancer (NSCLC). METHODS: The relative expressions of 11 miRNAs in sputum (miR-21, miR-145, miR-155, miR-205, miR-210, miR-92, miR-17-5p, miR-143, miR-182, miR-372, and let-7a) in addition to U6 were retrospectively assessed in four NSCLC-positive and four negative controls. Subsequently, a set of five miRNAs (miR-21, miR-143, miR-155, miR-210, miR-372) was selected because of degree of relatedness observed in the cluster analysis and tested in the same sputum sample set. The five optimized miRNAs accurately clustered these eight retrospective patients into NSCLC positive cases and negative controls. The five miRNA panel was then prospectively quantified in the sputum of 30 study patients (24 NSCLC cases and six negative controls) in a double-blind fashion to validate a five miRNA panel using hierarchical cluster analysis. RESULTS: The optimized five miRNA panel detected NSCLC (83.3% sensitivity and 100% specificity) in 30 prospectively accrued study patients. CONCLUSION: Sputum miRNA profiling using cluster analysis is a promising approach for the early detection of non-small cell lung cancer. Further investigation using this approach is warranted.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Detecção Precoce de Câncer/métodos , Perfilação da Expressão Gênica/métodos , Neoplasias Pulmonares/diagnóstico , MicroRNAs/genética , Escarro/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Método Duplo-Cego , Feminino , Humanos , Neoplasias Pulmonares/genética , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
The treatment of ovarian cancer has traditionally been intractable, and required novel approaches to improve therapeutic efficiency. This paper reports that thio-glucose bound gold nanoparticles (Glu-GNPs) can be used as a sensitizer to enhance ovarian cancer radiotherapy. The human ovarian cancer cells, SK-OV-3, were treated by gold nanoparticles (GNPs) alone, irradiation alone, or GNPs in addition to irradiation. Cell uptake was assayed using inductively coupled plasma atomic emission spectroscopy (ICP-AES), while cytotoxicity induced by radiotherapy was measured using both 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide and clonogenic assays. The presence of reactive oxygen species (ROS) was determined using CM-H2-DCFDA confocal microscopy and cell apoptosis was determined by an Annexin V-FITC/propidium iodide (PI) kit with flow cytometry. The cells treated by Glu-GNPs resulted in an approximate 31% increase in nanoparticle uptake compared to naked GNPs (p < 0.005). Compared to the irradiation alone treatment, the intracellular uptake of Glu-GNPs resulted in increased inhibition of cell proliferation by 30.48% for 90 kVp and 26.88% for 6 MV irradiation. The interaction of x-ray radiation with GNPs induced elevated levels of ROS production, which is one of the mechanisms by which GNPs can enhance radiotherapy on ovarian cancer.
Assuntos
Glucose/análogos & derivados , Glucose/toxicidade , Glucose/uso terapêutico , Ouro/toxicidade , Nanopartículas Metálicas/toxicidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/radioterapia , Compostos de Sulfidrila/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Fluorescência , Glucose/química , Ouro/química , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Neoplasias Ovarianas/metabolismo , Tolerância a Radiação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Compostos de Sulfidrila/química , Raios XRESUMO
Glucose-capped gold nanoparticles (Glu-GNPs) have been used to improve cellular targeting and radio-sensitization. In this study, we explored the mechanism of Glu-GNP enhanced radiation sensitivity in radiation-resistant human prostate cancer cells. Cell survival and proliferation were measured using MTT and clonogenic assay. Flow cytometry with staining by propidium iodide (PI) was performed to study the cell cycle changes induced by Glu-GNPs, and western blotting was used to determine the expression of p53 and cyclin proteins that correlated to cell cycle regulation. With 2 Gy of ortho-voltage irradiation, Glu-GNP showed a 1.5-2.0 fold enhancement in growth inhibition when compared to x-rays alone. Comparing the cell cycle change, Glu-GNPs induced acceleration in the G0/G1 phase and accumulation of cells in the G2/M phase at 29.8% versus 18.4% for controls at 24 h. G2/M arrest was accompanied by decreased expression of p53 and cyclin A, and increased expression of cyclin B1 and cyclin E. In conclusion, Glu-GNPs trigger activation of the CDK kinases leading to cell cycle acceleration in the G0/G1 phase and accumulation in the G2/M phase. This activation is accompanied by a striking sensitization to ionizing radiation, which may have clinical implications.
Assuntos
Ouro/química , Nanopartículas Metálicas/química , Neoplasias da Próstata/radioterapia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Citometria de Fluxo , Glucose/química , Glucose/farmacocinética , Glucose/farmacologia , Ouro/farmacocinética , Ouro/farmacologia , Humanos , MasculinoRESUMO
Gold nanoparticles (GNPs) and modified GNPs having two kinds of functional molecules, cysteamine (AET) and thioglucose (Glu), are synthesized. Cell uptake and radiation cytotoxicity enhancement in a breast-cancer cell line (MCF-7) versus a nonmalignant breast-cell line (MCF-10A) are studied. Transmission electron microscopy (TEM) results show that cancer cells take up functional Glu-GNPs significantly more than naked GNPs. The TEM results also indicate that AET-capped GNPs are mostly bound to the MCF-7 cell membrane, while Glu-GNPs enter the cells and are distributed in the cytoplasm. After MCF-7 cell uptake of Glu-GNPs, or binding of AET-GNPs, the in vitro cytotoxicity effects are observed at 24, 48, and 72 hours. The results show that these functional GNPs have little or no toxicity to these cells. To validate the enhanced killing effect on cancer cells, various forms of radiation are applied such as 200 kVp X-rays and gamma-rays, to the cells, both with and without functional GNPs. By comparison with irradiation alone, the results show that GNPs significantly enhance cancer killing.
Assuntos
Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Ouro/química , Ouro/farmacologia , Nanopartículas Metálicas/química , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ouro/metabolismo , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Sensibilidade e Especificidade , Análise EspectralRESUMO
PURPOSE: Nanotechnology is an emerging field with significant translational potential in medicine. In this study, we applied gold nanoparticles (GNP) to enhance radiation sensitivity and growth inhibition in radiation-resistant human prostate cancer cells. METHODS: Gold nanoparticles (GNPs) were synthesized using HAuCl4 as the gold particle source and NaBH4 as the reductant. Either thio-glucose or sodium citrate was then added to the solution separately to bind the GNPs to form thio-glucose-capped gold nanoparticles (Glu-GNP) and neutral gold nanoparticles (TGS-GNPs). Human prostate carcinoma DU-145 cells were exposed to vehicle, irradiation, 15nM TGS-GNPs, or 15nM Glu-GNPs, or GNPs plus irradiation. The uptake assays of GNP were performed using hemocytometer to count cells and the mass spectrometry was applied to calculate gold mass. The cytotoxicity induced by GNPs, irradiation, or GNPs plus irradiation was measured using a standard colorimetric MTT assay. RESULTS: Exposure to Glu-GNPs resulted in a three times increase of nanoparticle uptake compared to that of TGS-GNPs in each target cell (p < 0.005). Cytoplasmic intracellular uptake of both TGS-GNPs and Glu-GNPs resulted in a growth inhibition by 30.57% and 45.97% respectively, comparing to 15.88% induced by irradiation alone, in prostate cancer cells after exposure to the irradiation. Glu-GNPs showed a greater enhancement, 1.5 to 2 fold increases within 72 hours, on irradiation cytotoxicity compared to TGS-GNPs. Tumour killing, however, did not appear to correlate linearly with nanoparticle uptake concentrations. CONCLUSION: These results showed that functional glucose-bound gold nanoparticles enhanced radiation sensitivity and toxicity in prostate cancer cells. In vivo studies will be followed to verify our research findings.
Assuntos
Ouro/farmacologia , Nanopartículas Metálicas/efeitos da radiação , Neoplasias da Próstata/radioterapia , Tolerância a Radiação , Sobrevivência Celular/efeitos da radiação , Ouro/farmacocinética , Humanos , Masculino , Neoplasias da Próstata/patologiaRESUMO
PURPOSE: To develop a nanofiber hydrogel (NF-hydrogel) for sustained and controlled release of the recombinant receptor activator of NF-kB ligand; (RANKL) and to characterize the release kinetics and bioactivity of the released RANKL. METHODS: Various concentrations of fluorescently-labelled RANKL protein were added to NF-hydrogels, composed of Acetyl-(Arg-Ala-Asp-Ala)4-CONH2 [(RADA)4] of different concentrations, to investigate the resulting in vitro release rates. The nano-structures of NF-hydrogel, with and without RANKL, were determined using atomic force microscopy (AFM). Released RANKL was further analyzed for changes in secondary and tertiary structure using CD spectroscopy and fluorescent emission spectroscopy, respectively. Bioactivity of released RANKL protein was determined using NFATc1 gene expression and tartrate resistant acid phosphatase (TRAP) activity of osteoclast cells as biomarkers. RESULTS: NF-hydrogel concentration dependent sustained release of RANKL protein was measured at concentrations between 0.5 and 2%(w/v). NF-hydrogel at 2%(w/v) concentration exhibited a sustained and slow-release of RANKL protein up to 48h. Secondary and tertiary structure analyses confirmed no changes to the RANKL protein released from NF-hydrogel in comparison to native RANKL. The results of NFATc1 gene mRNA expression and TRAP activities of osteoclast, showed that the release process did not affect the bioactivity of released RANKL. CONCLUSIONS: This novel study is the first of its kind to attempt in vitro characterization of NF-hydrogel based delivery of RANKL protein to induce osteoclastogenesis. We have shown the self-assembling NF-hydrogel peptide system is amenable to the sustained and controlled release of RANKL locally; that could in turn increase local concentration of RANKL to induce osteoclastogenesis, for application to the controlled mobilization of tooth movement in orthodontic procedures. STATEMENT OF SIGNIFICANCE: Orthodontic tooth movement (OTM) occurs through controlled application of light forces to teeth, facilitating the required changes in the surrounding alveolar bone through the process of bone remodelling. The RANKL system regulates alveolar bone remodelling and controls root resorption during OTM. The use of exogenous RANKL to accelerate OTM has not been attempted to date because large quantities of RANKL for systemic therapy may subsequently cause serious systemic loss of skeletal bone. The controlled and sustained local release of RANKL from a carrier matrix could maximize its therapeutic benefit whilst minimizing systemic side effects. In this study a NF-hydrogel was used for sustained and controlled release of RANKL and the release kinetics and biofunctionality of the released RANKL was characterized. Our results provide fundamental insight for further investigating the role of RANKL NF-hydrogel release systems for inducing osteoclastogenesis in vivo.
Assuntos
Hidrogéis/farmacologia , Nanofibras/química , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Animais , Dicroísmo Circular , Liberação Controlada de Fármacos , Humanos , Cinética , Camundongos , Microscopia de Força Atômica , Nanofibras/ultraestrutura , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Estrutura Secundária de Proteína , Ligante RANK/química , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real , Fosfatase Ácida Resistente a Tartarato/metabolismoRESUMO
Vaccination is a safe and effective approach to prevent deadly diseases. To increase vaccine production, we propose that a mechanical stimulation can enhance protein production. In order to prove this hypothesis, Sf9 insect cells were used to evaluate the increase in the expression of a fusion protein from hepatitis B virus (HBV S1/S2). We discovered that the ultrasound stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm2, for a duration of 10 minutes per day increased HBV S1/S2 by 27%. We further derived a model for transport through a cell membrane under the effect of ultrasound waves, tested the key assumptions of the model through a molecular dynamics simulation package, NAMD (Nanoscale Molecular Dynamics program) and utilized CHARMM force field in a steered molecular dynamics environment. The results show that ultrasound waves can increase cell permeability, which, in turn, can enhance nutrient / waste exchange thus leading to enhanced vaccine production. This finding is very meaningful in either shortening vaccine production time, or increasing the yield of proteins for use as vaccines.
Assuntos
Vacinas contra Hepatite B/biossíntese , Ondas Ultrassônicas , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animais , Western Blotting , Permeabilidade da Membrana Celular , Desoxiglucose/análogos & derivados , Desoxiglucose/metabolismo , Vacinas contra Hepatite B/imunologia , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Proteínas/metabolismo , Células Sf9 , Sonicação , TermodinâmicaRESUMO
This study reports in-house assessment of a real-time cell electronic sensing (RT-CES) system used as a test platform for both cytotoxicity assay and predicting acute toxicity. For cytotoxicity determination, the RT-CES assay displayed equal sensitivity and coefficients of variation values with good correlation to NRU assay. The IC50 values and the LD50 values for the cytotoxicity reference materials were compared in the context of the proposed prediction model for acute rodent toxicity. The results obtained from RT-CES assay fitted within the acceptance limits of the prediction model and showed that the RT-CES cytotoxicity assay met the qualification guidelines in NIH Publication #01-4500 to accurately predict acute toxicity. In addition to cell viability, the RT-CES assay provided dynamic information that can be used to identify maximum toxicity and reversibility of the toxic effects which are difficult to achieve by the endpoint assays and, therefore, the RT-CES assay is more accurate for assessment of cytotoxicity. The features of the RT-CES assay, such as labeling free, automatic detection, and easy operation, give this assay potential to replace BALB/c 3T3 NRU assay and be used as routine setting for drug monitoring in the toxicological laboratory.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Testes de Toxicidade Aguda/métodos , Animais , Células 3T3 BALB , Contagem de Células , Camundongos , Microeletrodos , Vermelho Neutro , Sensibilidade e EspecificidadeRESUMO
A microelectronic array assay was developed to specifically genotype Helicobacter pylori versus Helicobacter heilmannii and to determine antimicrobial resistance. Helicobacter 16S rRNA and 23S rRNA genes were specifically generated with Helicobacter genus-specific primers, respectively. The single-nucleotide polymorphisms (SNPs) in 16S rRNA, 268T specific in the H. pylori sequence, and 263A specific in H. heilmannii were used as molecular markers for identification of H. pylori and H. heilmannii, respectively. A triple-base-pair resistant mutation, AGA965-967TTC in 16S rRNA, is known to be responsible for H. pylori tetracycline resistance and was detected to identify resistant strains. H. pylori macrolide resistance was determined by the identification of 3 defined mutations in the 23S rRNA gene using the same method. The assay could be directly used to detect H. pylori in feces. The assay performs multiple determinations, including identification of Helicobacter species and antibiotic resistances, on the same microelectronic platform and is highly amenable to the development of other DNA-based assays.
Assuntos
Helicobacter heilmannii/isolamento & purificação , Helicobacter pylori/isolamento & purificação , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Resistência a Tetraciclina , Sequência de Bases , Eletroforese , Fezes/microbiologia , Genótipo , Helicobacter heilmannii/efeitos dos fármacos , Helicobacter heilmannii/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Dados de Sequência Molecular , Mutação , Polimorfismo de Nucleotídeo Único , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Resistência a Tetraciclina/genéticaRESUMO
Arsenic is a pervasive cytotoxin and carcinogen in the environment. Although its mode of action has yet to be fully elucidated, oxidative DNA damage has been suggested. A series of DNA repair-defective human and hamster cell lines associated with sensitivity to oxidative agents were examined for their response to arsenic-induced cytotoxicity. Only the Ataxia telangiectasia (AT) cells displayed a marked hypersensitive response (greater than twofold). The protective role of the ATM protein was confirmed by the normal response to arsenic displayed by AT cells expressing wild-type ATM. Although the ATM protein plays a pivotal role in response to DNA double-strand breakage, none of the other cell lines with defects in double-strand break repair displayed a similar hypersensitivity. Further examination indicated that concentrations of sodium arsenite as high as 1 mg/l do not generate significant levels of double-strand breaks. Our data suggest that the ATM protein functions in an important but different capacity in the cellular response to arsenic toxicity than it does in response to agents that generate double-strand breaks, such as ionizing radiation. Furthermore, the lack of hypersensitivity to arsenic displayed by the other cell lines calls into question the hypothesis that DNA damage is a significant factor in arsenic cytotoxicity.
Assuntos
Arsenitos/toxicidade , Carcinógenos/toxicidade , Dano ao DNA , Proteínas Serina-Treonina Quinases/fisiologia , Compostos de Sódio/toxicidade , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Células CHO , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular , Linhagem Celular , Cricetinae , Reparo do DNA , Proteínas de Ligação a DNA , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Mutação , Neoplasias/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Hepatitis B is an infectious liver disease and vaccination is an effective way to protect individuals. We have applied mechanical wave stimulation to increase protein production. To validate our design, we used Sf9 insect cells to increase antigen fragment fusion protein expression for hepatitis B virus (HBV S1/S2). We discovered that stimulation at a frequency of 1.5 MHz, intensity of 60 mW/cm(2), for a duration of 10 minutes per day increased HBV S1/S2 production by 15%. This finding is very significant for shortening vaccine production time or increasing the yield of proteins for use as vaccines.
Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vacinas contra Hepatite B , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ondas Ultrassônicas , Animais , Linhagem Celular , Desenho de Equipamento , Antígenos de Superfície da Hepatite B/genética , Engenharia de Proteínas/instrumentação , Proteínas Recombinantes de Fusão/genéticaRESUMO
We previously described a sensitive assay for measuring thymine glycol in the DNA of irradiated cells. The assay combines immunorecognition of the DNA lesion with capillary electrophoresis and laser-fluorescence detection to achieve an absolute detection level in the zeptomole (10(-21) mol) range. This article describes modifications to the protocol that overcome certain technical problems seen with the original methodology. In particular, the capillary electrophoresis is carried out at pH 8.3 rather than pH 10.5. The new protocol was used to examine removal of thymine glycol from the DNA of A549 lung adenocarcinoma cells and resting lymphocytes after exposure to 2 Gy gamma rays. Both cell types displayed similar repair kinetics. Removal of thymine glycol is almost complete at 4 hours postirradiation.
Assuntos
Adenocarcinoma/genética , DNA/efeitos da radiação , Neoplasias Pulmonares/genética , Timidina/análogos & derivados , Eletroforese Capilar , Imunofluorescência , Humanos , Concentração de Íons de Hidrogênio , Timidina/efeitos da radiaçãoRESUMO
Aptamers are, in general, easier to produce, easier to store and are able to bind to a wider variety of targets than antibodies. For these reasons, aptamers are gaining increasing popularity in environmental monitoring as well as disease detection and disease management applications. This review article examines the research and design of RNA and DNA aptamer based biosensor systems and applications as well as their potential for integration in effective biosensor devices. As single stranded DNA or RNA molecules that can bind to specific targets, aptamers are well suited for biomolecular recognition and sensing applications. Beyond being able to be designed for a near endless number of specific targets, aptamers can also be made which change their conformation in a predictable and consistent way upon binding. This can lead to many unique and effective detection methods using a variety of optical and electrochemical means.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnica de Seleção de Aptâmeros , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas , Ouro , Nanopartículas Metálicas , Técnicas Analíticas Microfluídicas , Técnica de Seleção de Aptâmeros/instrumentação , Técnica de Seleção de Aptâmeros/métodosRESUMO
Many technologies, such as cell line screening and host cell engineering, culture media optimization and bioprocess optimization, have been proposed to increase monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. Unlike the existing biochemical approaches, we investigated stimulation using low-intensity pulsed ultrasound (LIPUS) as a purely physical approach, offering enhanced scalability, contamination control and cost-efficiency, while demonstrating significantly increased cell growth and antibody production. It was found that daily ultrasound treatments at 40 mW/cm(2) for 5 min during cell culture increased the production of human anti-IL-8 antibody by more than 30% using 10 or 30 mL shake flasks. Further increasing the ultrasound dosage (either intensities or the treatment duration) did not appreciably increase cell growth or antibody production, however feeding the culture with additional highly-concentrated nutrients, glucose and amino acids (glutamine in this case), did further increase cell growth and antibody titer to 35%. Similar ultrasound treatments (40 mW/cm(2), 5 min per day) when scaled up to larger volume wavebags, resulted in a 25% increase in antibody production. Increased antibody production can be attributed to both elevated cell count and the ultrasound stimulation. Theoretical study of underlying mechanisms was performed through the simulations of molecular dynamics using the AMBER software package, with results showing that LIPUS increases cell permeability. The significance of this study is that LIPUS, as a physical-based stimulation approach, can be externally applied to the cell culture without worrying about contamination. By combining with the existing technologies in antibody production, LIPUS can achieve additional mAb yields. Because it can be easily integrated with existing cell culture apparatuses, the technology is expected to be more acceptable by the end users.
Assuntos
Anticorpos Monoclonais/biossíntese , Reatores Biológicos , Células CHO/diagnóstico por imagem , Células CHO/metabolismo , Sonicação/métodos , Animais , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular , Cricetulus , Ensaio de Imunoadsorção Enzimática , Engenharia de Proteínas/métodos , Sonicação/instrumentação , Transdutores , UltrassonografiaRESUMO
Pharmacokinetics and bio-distribution are crucial factors affecting the performance of an intravenous drug. In this study, we explore the combined use of glucose and polyethylene glycol (PEG) ligands to further improve gold nanoparticle (GNP) pharmacokinetics and bio-distribution, with the aim of using the drug for in-vivo radiotherapy. The inclusion of PEG was found to significantly prolong the half-life period, where PEG-Glu-GNPs achieved 6.17 +/- 3.71 h, compared to 1.23 +/- 0.14 h for Glu-GNPs and 1.07 +/- 0.22 h for uncoated GNPs. Our data indicates that nanoparticle size impacts cell uptake performance, with 20 nm being the optimal diameter for cancer treatment applications. Although PEG-Glu-GNPs mainly distributed in the spleen, liver, lung, and kidneys, the concentration of PEG-Glu-GNPs in tumour tissue was 20 times higher than healthy cells in the uterus and ovaries, reaching 9.22 +/- 2.41 microg/g cancer tissue at 48 h after injection. This difference in uptake holds promise for selective tumor targeting which can in turn lead to more effective radiotherapy through the interaction of X-rays and GNPs. Specifically tumor size after 47 days of treatment had reduced to (769 +/- 92) mm3 compared to (1432 +/- 269) mm3 using X-rays alone and (3514 +/- 1818) mm3 without any treatment. Moreover, the mice remained healthy without statistically significant weight loss. Results of our pharmacokinetic and bio-distribution study as well as therapeutic data for PEG-Glu-GNPs in our tumor bearing animal model demonstrate that PEG-Glu-GNPs provide excellent in-vivo stability, tumor targeting function, and radiotherapeutic enhancement effects, providing useful insights for further clinical studies.
Assuntos
Ouro/farmacocinética , Nanopartículas Metálicas/química , Polietilenoglicóis/farmacocinética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/radioterapia , Animais , Linhagem Celular Tumoral , Feminino , Ouro/farmacologia , Humanos , Nanopartículas Metálicas/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Polietilenoglicóis/farmacologia , Tomografia por Emissão de Pósitrons , Ratos , Ratos Wistar , Distribuição Tecidual , Neoplasias do Colo do Útero/diagnóstico por imagem , Neoplasias do Colo do Útero/ultraestruturaRESUMO
UNLABELLED: Paclitaxel is a microtubule inhibitor causing mitotic arrest and is widely used in cancer chemotherapy. However, its poor water solubility restricts its direct clinical applications. In this article, we report paclitaxel-loaded nanoparticles that are water soluble and that can improve the drug's bio-distribution and therapeutic efficacy. Paclitaxel-loaded nanoparticles were synthesized by using Pluronic copolymers (F-68 and P-123) and surfactant (Span 40) as nanocarrier. The toxicity and cellular uptake of paclitaxel-loaded nanoparticles were evaluated. The paclitaxel-loaded nanoparticles can completely disperse into phosphate buffer saline to produce a clear aqueous suspension. Based on HPLC analysis, the drug-loading rate is 9.0 ± 0.1% while drug encapsulation efficiency is 99.0 ± 1.0%. The cytotoxicity assay was performed using breast cancer MCF-7 and cervical cancer Hela cells. For MCF-7 cells, the half maximal inhibitory concentrations (IC50) of paclitaxel-loaded nanoparticles and paclitaxel are 8.5 ± 0.3 and 14.0 ± 0.7 ng/mL at 48 hours and 3.5 ± 0.4 and 5.2 ± 0.5 ng/mL at 72 hours across several runs. IC50 of paclitaxel-loaded nanoparticles and paclitaxel for Hela cells are 5.0 ± 0.3 and 8.0 ± 0.3 ng/mL at 48 hours and 2.0 ± 0.1 and 6.5 ± 0.3 ng/mL at 72 hours. In-vitro studies show that the drug's nanoformulation gives obvious enhancements in the drug's efficiency at killing cancer cells over paclitaxel alone. Materials of the nanocarrier used for nanoformulation are approved with low toxicity according to the result of cell studies. CONCLUSION: paclitaxel-loaded nanoparticles greatly improved the physicochemical properties of paclitaxel without modifying its chemical structure, allowing for deep-site cancer drug delivery and enhancing the drug therapeutic efficiency.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Portadores de Fármacos/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Poloxâmero/química , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Paclitaxel/farmacologiaRESUMO
With the rapidly growing demand for monoclonal antibody (mAb)-based products, new technologies are urgently needed to increase mAb production while reducing manufacturing costs. To solve this problem, we report our research findings of using low-intensity pulsed ultrasound (LIPUS) to enhance mAb production. LIPUS with frequency of 1.5 MHz and pulse repetition frequency of 1 kHz, as well as duty cycle of 20%, was used to stimulate hybridoma cells to enhance the production of mAb, anti-CD4 (hybridoma GK1.5). The enzyme-linked immunosorbent assay results show a 60.42 ± 7.63% increase of mAb expression in hybridoma cells. The evidence of structural changes of the cellular outer membrane in both transmission electron microscopy and scanning electron microscopy images and the more than 20% lactate dehydrogenase release indicates that the increased mAb production is related to the increased cell permeability induced by LIPUS. This value-added ultrasound technology provides a potential cost-effective solution for pharmaceutical companies to manufacture mAb-based drugs. The technology, in turn, can reduce the drug manufacturing costs and decrease health care spending.