RESUMO
Particularly, chitosan (Cs) loaded with drug cannot pass through the colonic region, often leading in the bursting drug release in the stomach due to its solubility in gastric contents. The novelty of the current article is to solve this limitation by performing gamma irradiation cross-linking of Cs with two anionic polymers of (acrylic acid)-co-(2-acrylamido-2-methylpropane-sulfonic acid) (AAc/AMPS) to give amphiphilic hydrogel. The shifted in the characteristic FTIR peaks of Cs in the (Cs/AAc/AMPS) confirm the exits of inter-molecular interactions that make Cs and (AAc/AMPS) are miscible. Swelling experiments under different pH indicated that the (Cs/AAc/AMPS) hydrogels were significantly sensitive to pH change. The results give the possibility to use the obtained (Cs/AAc/AMPS) hydrogel on drug delivery system. The in vitro Fluorouracil (5-FU) releasing from (Cs/AAc/AMPS) matrix was examined under the influence of pH1 and pH7.The results confirmed the hydrogels capability to release 96 % of 5-FU drug at pH 7 after 7 h.
Assuntos
Quitosana/síntese química , Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Raios gama , Hidrogéis/síntese química , Polímeros/síntese química , Tensoativos/síntese química , Acrilamidas/química , Acrilatos/química , Alcanossulfonatos/química , Antimetabólitos Antineoplásicos/química , Quitosana/química , Reagentes de Ligações Cruzadas , Liberação Controlada de Fármacos , Fluoruracila/química , Humanos , Hidrogéis/química , Concentração de Íons de Hidrogênio , Cinética , Polímeros/química , Tensoativos/química , Água/químicaRESUMO
BACKGROUND: In Doxil®, PEGylated nanoliposomes are created by hydration of the lipids in ammonium sulfate, and are remotely loaded with doxorubicin by a transmembrane ammonium gradient. The ammonium sulfate is then removed from the external aqueous phase, surrounding the liposomes, and replaced by an isoosmotic sucrose solution in 10 mM histidine buffer at pH 6.5. METHODS: We prepared PEGylated liposomal doxorubicin (PLD) with a series of ammonium monovalent salts that after remote loading became the intraliposome doxorubicin counteranions. We analyzed the liposomes by solution X-ray scattering, differential scanning calorimetry, and electron micropscopy. RESULTS: PLDs prepared with sulfonic acid derivatives as counteranion exhibited chemical and physical stabilities. We determined the effect of these ammonium salt counteranions on the structure, morphology, and thermotropic behavior of the PEGylated nanoliposomes, formed before and after doxorubicin loading, and the bulk properties of the doxorubicin-counteranion complexes. By comparing the structure of the doxorubicin complexes in the bulk and inside the nanoliposomes, we revealed the effect of confinement on the structure and doxorubicin release rate for each of the derivatives of the ammonium sulfonic acid counteranions. CONCLUSIONS: We found that the extent and direction of the doxorubicin confinement effect and its release rate were strongly dependent on the type of counteranion. The counteranions, however, neither affected the structure and thermotropic behavior of the liposome membrane, nor the thickness and density of the liposome PEG layers. In an additional study, it was demonstrated that PLD made with ammonium-methane sulfonate exhibit a much lower Hand and Foot syndrome. GENERAL SIGNIFICANCE: The structure, physical state, and pharmacokinetics of doxorubicin in PEGylated nanoliposomes, prepared by transmembrane remote loading using gradients of ammonium salts, strongly depend on the counteranions.
Assuntos
Compostos de Amônio/química , Antibióticos Antineoplásicos/química , Doxorrubicina/análogos & derivados , Polietilenoglicóis/química , Alcanossulfonatos/química , Sulfato de Amônio/química , Ânions/química , Cristalização , Doxorrubicina/química , Mesilatos/químicaRESUMO
Here, firstly lignin sulfonate was produced from sulfite liquor provided by Mazandaran wood & paper industries (Chookam). Then, the role of effective parameters including reaction temperature, duration time, and amounts of pyridine and acetic anhydride respectively as the catalyst and the esterification agent on the acetylation rate of lignin sulfonate were studied and the process parameters were optimized through multiple experiments. In this investigation, using 1 g lignin sulfonate, the effect of various levels of temperature (ranged from room temperature to 140 °C), reaction time (12-72 h), pyridine volume (0-30 mL), and acetic anhydride volume (5-30 mL) were evaluated. Based on the results of several esterification processes, the optimal values of temperature and reaction time were obtained to be 100 °C and 48 h, respectively, and the optimal volumes of acetic anhydride and pyridine were 20 mL (with equal amounts). Besides, the characterization tests of lignin sulfonate and acetylated lignin sulfonate were performed using FT-IR and NMR techniques. Also in this paper, the morphology and crystallinity/amorphicity of lignin sulfonate and acetylated lignin sulfonate were examined using SEM images and XRD patterns.
Assuntos
Alcanossulfonatos/química , Biopolímeros/química , Lignina/química , Sulfitos/química , Anidridos Acéticos/química , Acetilação , Esterificação , Lignina/síntese química , Espectroscopia de Ressonância Magnética , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Madeira/químicaRESUMO
OBJECTIVE: PPARα/γ dual agonists have been in clinical development for the treatment of metabolic diseases including type 2 diabetes and dyslipidemia. However, severe adverse side effects led to complications in clinical trials. As most of the beneficial effects rely on the compound activity in adipocytes, the selective targeting of this cell type is a cutting-edge strategy to develop safe anti-diabetic drugs. The goal of this study was to strengthen the adipocyte-specific uptake of the PPARα/γ agonist tesaglitazar via NPY1R-mediated internalization. METHODS: NPY1R-preferring peptide tesaglitazar-[F7, P34]-NPY (tesa-NPY) was synthesized by a combination of automated SPPS and manual couplings. Following molecular and functional analyses for proof of concept, cell culture experiments were conducted to monitor the effects on adipogenesis. Mice treated with peptide drug conjugates or vehicle either by gavage or intraperitoneal injection were characterized phenotypically and metabolically. Histological analysis and transcriptional profiling of the adipose tissue were performed. RESULTS: In vitro studies revealed that the tesaglitazar-[F7, P34]-NPY conjugate selectively activates PPARγ in NPY1R-expressing cells and enhances adipocyte differentiation and adiponectin expression in adipocyte precursor cells. In vivo studies using db/db mice demonstrated that the anti-diabetic activity of the peptide conjugate is as efficient as that of systemically administered tesaglitazar. Additionally, tesa-NPY induces adipocyte differentiation in vivo. CONCLUSIONS: The use of the tesaglitazar-[F7, P34]-NPY conjugate is a promising strategy to apply the beneficial PPARα/γ effects in adipocytes while potentially omitting adverse effects in other tissues.
Assuntos
Adipócitos/efeitos dos fármacos , Alcanossulfonatos/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 2/prevenção & controle , Peptídeos/farmacologia , Fenilpropionatos/farmacologia , Receptores de Neuropeptídeo Y/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Alcanossulfonatos/administração & dosagem , Alcanossulfonatos/química , Animais , Células COS , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Peptídeos/administração & dosagem , Peptídeos/química , Fenilpropionatos/administração & dosagem , Fenilpropionatos/química , Relação Estrutura-AtividadeRESUMO
A series of hybrid proton exchange membranes were synthesized via in situ polymerization of poly (2-acrylamido-2-methyl-1-propanesulfonic acid) PMPS with sulfonated poly (1,4-phenylene ether-ether-sulfone) (SPEES). The insertion of poly (2-acrylamido-2-methyl-1-propanesulfonic acid) PMPS, between the rigid skeleton of SPEES plays a reinforcing role to enhance the ionic conductivity. The synthesized polymer was chemically characterized by fourier-transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance 1H NMR spectroscopy to demonstrate the successful grafting of PMPS with the pendent polymer chain of SPEES. A variety of physicochemical properties were also investigated such as ion exchange capacity (IEC), proton conductivity, water uptake and swelling ratio to characterize the suitability of the formed polymer for various electrochemical applications. SP-PMPS-03, having the highest concentration of all PMPS, shows excellent proton conductivity of 0.089 S cm-1 at 80 °C which is much higher than SPEES which is ~0.049 S cm-1. Optimum water uptake and swelling ratio with high conductivity is mainly attributed to a less ordered arrangement polymer chain with high density of the functional group to facilitate ionic transport. The residual weight was 93.35, 92.44 and 89.56%, for SP-PMPS-01, 02 and 03, respectively, in tests with Fenton's reagent after 24 h. In support of all above properties a good chemical and thermal stability was also achieved by SP-PMPS-03, owing to the durability for electrochemical application.
Assuntos
Acrilamidas/química , Alcanossulfonatos/química , Condutividade Elétrica , Substâncias Intercalantes/química , Membranas Artificiais , Polímeros/química , Prótons , Sulfonas/química , ÍonsRESUMO
Alkylaryl sulfonate is a typical family of surfactants used for chemically enhanced oil recovery (EOR). While it has been widely used in surfactant-polymer flooding at Karamay Oilfield (40 °C, salinity 14,000 mg/L), its aggregation behavior in aqueous solutions and the contribution of aggregation to EOR have not been investigated so far. In this study, raw naphthenic arylsulfonate (NAS) and its purified derivatives, alkylaryl monosulfonate (AMS) and alkylaryl disulfonate (ADS), were examined under simulated temperature and salinity environment of Karamay reservoirs for their micellar aggregation behavior through measuring surface tension, micellar size, and micellar aggregation number. It was found that all three alkylaryl sulfonate surfactants could significantly lower the surface tension of their aqueous solutions. Also, it has been noted that an elevation both in temperature and salinity reduced the surface tension and critical micellar concentration. The results promote understanding of the performance of NAS and screening surfactants in EOR.
Assuntos
Alcanossulfonatos/química , Óleos/química , Tensoativos/química , Micelas , Tensão Superficial , Temperatura , Água/químicaRESUMO
Benefiting from the simple DNAzyme and a duplex-specific nuclease, a series of microRNA-stimulated DNAzyme logic gates were rationally assembled for the highly accurate detection of multiple low-abundant microRNA biomarkers that correspond to the specific aberrant microRNA expression patterns of cancer tissues, thus showing a great potential in early diagnosis.
Assuntos
Biomarcadores Tumorais/análise , Computadores Moleculares , DNA Catalítico/química , MicroRNAs/análise , Neoplasias/diagnóstico , Alcanossulfonatos/química , Compostos Azo/química , Endodesoxirribonucleases/química , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodosRESUMO
Platelet-derived growth factor-BB (PDGF-BB) is currently used as a biomarker protein for cancer early diagnosis and clinical treatment. Herein, we reported a robust and enzyme-free strategy based on aptamer recognition and proximity-induced entropy-driven circuits (AR-PEDC) for homogeneous and rapid detection of platelet-derived growth factor BB (PDGF-BB) without any washing steps or thermocycling. The proximity probes specifically recognize target protein to form the completed trigger (CT). Then, the CT reacts with three-strand complex to form intermediate, which subsequently binds to fuel strand to release reporter strand, assistant strand and the CT. The revised proximity probes exhibit significantly improved signal-to-background ratio and faster association rate. Moreover, target protein/proximity probes interaction can specifically initiate entropy-driven circuits, thus providing immense signal amplification for ultrasensitive detection of PDGF-BB with low detection limit of 9.6 pM. The practical ability of the developed strategy is demonstrated by detection of PDGF-BB in human serum with satisfactory results. In addition, this method is flexible and can be conveniently extended to a variety of targets by simply substituting the target specific sequence. Thus, this strategy presents a rapid, low background and versatile amplification mechanism for the detection of protein biomarkers and offers a promising alternative platform for clinical diagnosis.
Assuntos
Aptâmeros de Nucleotídeos/química , Becaplermina/sangue , Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/métodos , Alcanossulfonatos/química , Aptâmeros de Nucleotídeos/genética , Compostos Azo/química , DNA/química , DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Espectrometria de Fluorescência/métodosRESUMO
Ferroptosis is an iron-dependent cell death caused by accumulation of lipid peroxidation (LPO), which is a new strategy for cancer treatment. Th current ferroptosis therapy nanodevices have low efficiency and side effects generally. Hence, we developed a Black Hole Quencher (BHQ)-based fluorescence "off-on" nanophotosensitizer complex assembly (CSO-BHQ-IR780-Hex/MIONPs/Sor). CSO-connected BHQ-IR780-Hex and -loaded magnetic iron oxide nanoparticles (MIONPs) and sorafenib (Sor) formed a very concise functionalized delivery system. CSO-BHQ-IR780-Hex disassembled by GSH attack and released IR780-Hex, MIONPs, and sorafenib. IR780-Hex anchored to the mitochondrial membrane, which would contribute to amplifying the efficiency of the photosensitizer. When NIR irradiation was given to CSO-BHQ-IR780-Hex/MIONPs/Sor-treated cells, iron supply increased, the xCT/GSH/GPX-4 system was triggered, and a lot of LPO burst. A malondialdehyde test showed that LPO in complex assembly-treated cells was explosive and increased about 18-fold compared to the control. The accumulation process of particles was monitored by an IR780-Hex photosensitizer, which showed an excellent tumor target ability by magnetic of nanodevice in vivo. Interestingly, the half-life of sorafenib in a nanodevice was increased about 26-fold compared to the control group. Importantly, the complex assembly effectively inhibits tumor growth in the breast tumor mouse model. This work would provide ideas in designing nanomedicines for the ferroptosis treatment of cancer.
Assuntos
Alcanossulfonatos , Compostos Azo , Neoplasias da Mama , Ferroptose/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Nanopartículas de Magnetita , Sorafenibe , Alcanossulfonatos/química , Alcanossulfonatos/farmacologia , Animais , Compostos Azo/química , Compostos Azo/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Sorafenibe/química , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-18F-fluoropropane-1-thiol and 2-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (18F-fluoro-PEG4 thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix222/K2CO3 conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-18F-fluoropropyl) benzothioate and 18F-fluoro-PEG4 benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15â¯min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]2), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting 18F-fluoride ranged from 6 to 22% in a synthesis time of about 90â¯min. 18F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]2 maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. 18F-fluoro-PEG4-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to 18F-fluoropropyl-S-E[c(RGDfK)]2. Bone uptake was low for both indicating in vivo stability.
Assuntos
Alcanossulfonatos/química , Indicadores e Reagentes/química , Maleimidas/farmacologia , Peptídeos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Compostos de Sulfidrila/química , Animais , Linhagem Celular Tumoral , Radioisótopos de Flúor/química , Humanos , Marcação por Isótopo/métodos , Maleimidas/síntese química , Camundongos Nus , Peptídeos/síntese química , Compostos Radiofarmacêuticos/síntese químicaRESUMO
We have formulated a rapid and high-efficiency fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced exponential rolling circular amplification (RCA) strategy for the ultrasensitive detection of miR-21. In this strategy, there are several aspects that are worthwhile. First, the functionalized THMS, comprising a typical triplex structure (T-A·T), specific recognition sequence for nicking endonuclease, complementary sequence for miR-21, and RCA product-annealing sequence, was concurrently used to perform signal transduction with one fluorophore and one quencher. As compared to the traditional double-helix molecular switches or molecular beacons, one of the biggest differentiating factors is that the properties of THMSs are independent of any specific binding sequence that they may contain. As far as we know, this is the first time that an ingeniously designed THMS not only contains the primer for exponential RCA, but also functions as the tracer for fluorescence assay. In the presence of miR-21, targets can induce conformation changes in THMS with the release of the trapped DNA segment (P), which, in turn, can activate the first run of the RCA process. Meanwhile, the RCA reaction is also initiated by the formation of a similar primer (SP) as the trapped DNA through a continuous "extension-nicking" reaction. Secondly, the resultant first-generation RCA product consists of numerous tandem repeated regions that can attach to countless THMSs, resulting in the release of the trapped DNA segment (P) for initiating the second run of the RCA reaction. Significantly, a large amount of THMSs were continuously consumed to yield a remarkably strong fluorescent signal. In addition, this biosensor was demonstrated to exhibit improved sensitivity owing to the high efficiency and rapid amplification kinetics of the exponential RCA and high selectivity toward miR-21 with a limit of detection as low as 1.1 aM. Hence, the target-mediated THMS-conformation-change-initiated exponential RCA strategy presents an optimal detection performance toward analytes for potential applications in related fundamental research and clinical diagnosis.
Assuntos
Técnicas Biossensoriais/métodos , MicroRNAs/análise , Alcanossulfonatos/química , Compostos Azo/química , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodosRESUMO
OBJECTIVE: The study describes the development of chitosan-based (AMPS-co-AA) semi-IPN hydrogels using free radical polymerization technique. METHODS: The resulting hydrogels were characterized using Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC), X-Ray diffraction (XRD), and Scanning Electron Microscopy (SEM). The successful crosslinking of chitosan, 2- Acrylamido-2-Methylpropane Sulfonic Acid (AMPS), and Acrylic Acid (AA) was confirmed by FT IR. Unloaded and drug-loaded hydrogels exhibited higher thermal stability after crosslinking compared to the individual components. XRD confirmed the decrease in crystallinity after hydrogel formation and molecular dispersion of Oxaliplatin (OXP) in the polymeric network. SEM showed rough, vague and nebulous surface resulting from crosslinking and loading of OXP. RESULTS: The experimental results revealed that swelling and drug release were influenced by the pH of the medium being low at acidic pH and higher at basic pH. Increasing the concentration of chitosan and AA enhanced the swelling, drug loading and drug release while AMPS was found to act inversely. CONCLUSION: It was confirmed that the hydrogels were degraded more by specific enzyme lysozyme as compared to the non-specific enzyme collagenase. In-vitro cytotoxicity suggested that the unloaded hydrogels were non-cytotoxic while crude drug and drug-loaded hydrogel exhibited dose-dependent cytotoxicity against HCT-116 and MCF-7. Results of acute oral toxicity on rabbits demonstrated that the hydrogels are non-toxic up to 3900 mg/kg after oral administration, as no toxicity or histopathological changes were observed in comparison to control rabbits. These pH-sensitive hydrogels appear to provide an ideal basis as a safe carrier for oral drug delivery.
Assuntos
Acrilamidas/administração & dosagem , Alcanossulfonatos/administração & dosagem , Quitosana/administração & dosagem , Portadores de Fármacos/administração & dosagem , Hidrogéis/administração & dosagem , Acrilamidas/química , Alcanossulfonatos/química , Animais , Quitosana/química , Colagenases/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Feminino , Células HCT116 , Humanos , Hidrogéis/química , Células MCF-7 , Masculino , Muramidase/química , Coelhos , Testes de Toxicidade AgudaRESUMO
Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn2+-specific near-infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photodecages a substrate strand containing a nitrobenzyl group at the 2'-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn2+-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn2+ sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos and detecting in the visible region. In this study, we introduce a platform that can be used to understand the Zn2+ distribution with spatiotemporal control, thereby giving insights into the dynamical Zn2+ ion distribution in intracellular and in vivo models.
Assuntos
DNA Catalítico/química , Corantes Fluorescentes/química , Nanopartículas/química , Zinco/análise , Alcanossulfonatos/química , Alcanossulfonatos/toxicidade , Animais , Compostos Azo/química , Compostos Azo/toxicidade , Sequência de Bases , DNA Catalítico/síntese química , DNA Catalítico/toxicidade , Fluoresceínas/química , Fluoresceínas/toxicidade , Fluorescência , Corantes Fluorescentes/toxicidade , Fluoretos/química , Fluoretos/toxicidade , Células HeLa , Humanos , Raios Infravermelhos , Microscopia Confocal , Microscopia de Fluorescência , Nanopartículas/efeitos da radiação , Nanopartículas/toxicidade , Túlio/química , Túlio/toxicidade , Itérbio/química , Itérbio/toxicidade , Ítrio/química , Ítrio/toxicidade , Peixe-ZebraRESUMO
Triplex molecular beacons (tMBs) possess great potential in biological sensing because of the pH responsiveness and controllability of binding strength. Here, we systematically investigate and rationally design a classical tMB for convenient detection of microRNA-21, a well-known biomarker of cardio-cerebrovascular diseases. In the tMB, we employ the complementary sequence of miR-21 as the loop and the sequences of protonated cytosine-guanine-cytosine (C-Gâ¢C+) and thymine-adenine-thymine (T-Aâ¢T) as the triplex stem, in which both the Watson-Crick and Hoogsteen base-pairing control the binding strength in cooperation. It is demonstrated for the first time that the presence of miR-21 would only break the Hoogsteen base-pairing in the stem and hybridize with the tMB to form the rigid heterozygous hybrid duplex structure. These would hinder the fluorescence resonance energy transfer (FRET) between the fluorophore (FAM) and quencher (BHQ1) labeled at the ends of the oligonucleotide, and the fluorescence recovery degree of FAM can be used as the standard to quantitate the miR-21. More significantly, the excellent adjustability and sensitivity of our tMBs have been confirmed by constructing the corresponding duplex molecular beacon (dMB) for comparison. The fluorophore FAM in the tMB could be replaced by the fluorescent DNA/silver nanoclusters, which exhibits the universal applicability of energy donor and receptor selection for tMB. Furthermore, our proposed tMB could also be developed as an aptasensor for the detection of vascular endothelial growth factor (VEGF) by only introducing the complementary sequence of its aptamer into the tMB. This work is of great significance for the systematic study of tMBs for the detection of biomarkers such as nucleic acids and proteins.
Assuntos
DNA/química , MicroRNAs/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Alcanossulfonatos/química , Compostos Azo/química , Pareamento de Bases , Sequência de Bases , DNA/genética , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genéticaRESUMO
In this study, a rapid, sensitive, and specific assay for detecting miRNAs was developed based on a two-stage amplification reaction (TSAR) using molecular beacons (MBs) as turn-on probes. In the TSAR, different miRNAs can be converted to the same reporter oligonucleotides (Y), which can hybridize with the same MB. Therefore, in combination with specific templates, this method can be applied to multiplex miRNA detection by simply using the same MB. The loop region of the MB was screened by computer simulation methods. In particular, to improve the specificity of the MB in real sample analysis, the maximum similarity of the MB loop region to the human genome and human transcriptome is less than 70%. Two MBs were designed in this study. MB I, with nine flanking base pairs in its stem region, was used for real-time monitoring of the production of Y during the TSAR. MB II, with five flanking base pairs in its stem region, was used to detect the production of Y at the end of the TSAR. This assay exhibited high sensitivity with a limit of detection of 2.0 × 10-16M and 6.7 × 10-16M using MB I and MB II as turn-on probes, respectively. In addition, this assay can clearly discriminate single base differences in miRNA sequences, and the TSAR can be completed under isothermal conditions. Accordingly, the isothermal reaction conditions and simple fluorescence measurement can greatly contribute to the development of a fast point-of-care detection system.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico , Alcanossulfonatos/química , Compostos Azo/química , Pareamento de Bases , Neoplasias do Colo/sangue , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/patologia , Sondas de DNA/síntese química , Sondas de DNA/química , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/sangue , Conformação de Ácido Nucleico , Reprodutibilidade dos TestesRESUMO
Hydrogel particles have proven to be powerful tools for the capture and concentration of low abundance, low molecular weight peptides and proteins from complex biofluids, such as plasma. The primary means of recovering and washing the particles following harvesting is through centrifugation, which can be a very time-consuming process depending on harvest conditions. To improve the process of particle recovery, washing, and elution we have developed new particle formulations: incorporating N-t-butylacrylamide (tBA) in the polymer backbone with monomers bearing more acidic functional groups and higher degrees of cross-linking. These particle formulations produce a stable architecture that does not significantly respond to changes in environmental conditions, such as pH and temperature. These two new formulations impart structural stability to the particle, control swelling, and improve pelleting through centrifugation, even at high pH values. These structurally stable microparticles yield improved particle recovery while maintaining the peptide capture properties of the particle.
Assuntos
Acrilamidas/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Peptídeos/química , Acrilatos/química , Alcanossulfonatos/química , Composição de Medicamentos , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Varredura , Tamanho da PartículaRESUMO
As an alternative to perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluorinated ether sulfonate (commercial name: F-53B) has been used as a mist suppressant in Chinese electroplating industries for over 30 years. It has been found in the environment and fish, and one acute assay indicated F-53B was moderately toxic. However, the toxicological information on this compound was incomplete and insufficient for assessment of their environment impact. The object of this study was to examine the developmental toxicity of F-53B using zebrafish embryos. Zebrafish embryos were incubated in 6-well plates with various concentrations of F-53B (1.5, 3, 6, and 12mg/L) from 6 to 132h post fertilization (hpf). Results showed that F-53B exposure induced developmental toxicity, including delayed hatching, increased occurrence of malformations, and reduced survival. Malformations, including pericardial and yolk sac edemas, abnormal spines, bent tails, and uninflated swim bladders, appeared at 84 hpf, and increased with time course and dose. A decrease in survival percentages was noted in the 6 and 12mg/L F-53B-treated groups at 132 hpf. Continuous exposure to 3mg/L F-53B resulted in high accumulation levels in zebrafish embryos, suggesting an inability for embryos to eliminate this compound and a high cumulative risk to fish. We also examined the cardiac function of embryos at specific developmental stages following exposure to different concentrations, and found that F-53B induced cardiac toxicity and reduced heart rate. Even under low F-53B concentration, o-dianisidine staining results showed significant decrease of relative erythrocyte number at 72 hpf before the appearance of observed effects of F-53B on the heart. To elucidate the underlying molecular changes, genes involved in normal cardiac development were analyzed using real-time qPCR in the whole-body of zebrafish embryos. F-53B inhibited the mRNA expression of ß-catenin (ctnnb2) and wnt3a. The mRNA levels of ß-catenin targeted genes (nkx2.5 and sox9b), which play critical roles in cardiogenesis, were also reduced after exposure. Thus, exposure to F-53B impaired the development of zebrafish embryos and disrupted cardiac development, which might be mediated by effects on the Wnt signaling pathway and decrease of erythrocyte numbers.
Assuntos
Alcanossulfonatos/toxicidade , Ácidos Alcanossulfônicos/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Fluorocarbonos/toxicidade , Coração/embriologia , Peixe-Zebra/embriologia , Alcanossulfonatos/química , Ácidos Alcanossulfônicos/química , Animais , Embrião não Mamífero/metabolismo , Eritrócitos/efeitos dos fármacos , Fluorocarbonos/química , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Coração/efeitos dos fármacos , Larva/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
DNA-alkylating drugs continue to remain an important weapon in the arsenal against cancers. However, they typically suffer from several shortcomings because of the indiscriminate DNA damage that they cause and their inability to specifically target cancer cells. We have developed a strategy for overcoming the deficiencies in current DNA-alkylating chemotherapy drugs by designing a site-specific DNA-methylating agent that can target cancer cells because of its selective uptake via glucose transporters, which are overexpressed in most cancers. The design features of the molecule, its synthesis, its reactivity with DNA, and its toxicity in human glioblastoma cells are reported here. In this molecule, a glucosamine unit, which can facilitate uptake via glucose transporters, is conjugated to one end of a bispyrrole triamide unit, which is known to bind to the minor groove of DNA at A/T-rich regions. A methyl sulfonate moiety is tethered to the other end of the bispyrrole unit to serve as a DNA-methylating agent. This molecule produces exclusively N3-methyladenine adducts upon reaction with DNA and is an order of magnitude more toxic to treatment resistant human glioblastoma cells than streptozotocin is, a Food and Drug Administration-approved, glycoconjugated DNA-methylating drug. Cellular uptake studies using a fluorescent analogue of our molecule provide evidence of uptake via glucose transporters and localization within the nucleus of cells. These results demonstrate the feasibility of our strategy for developing more potent anticancer chemotherapeutics, while minimizing common side effects resulting from off-target damage.
Assuntos
Antineoplásicos Alquilantes/síntese química , Adutos de DNA/biossíntese , DNA de Neoplasias/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glicoconjugados/síntese química , Neuroglia/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Alcanossulfonatos/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/farmacologia , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/química , Dano ao DNA , Metilação de DNA , DNA de Neoplasias/química , DNA de Neoplasias/metabolismo , Expressão Gênica , Glucosamina/química , Proteínas Facilitadoras de Transporte de Glucose/genética , Glicoconjugados/metabolismo , Glicoconjugados/farmacologia , Humanos , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Neuroglia/metabolismo , Neuroglia/patologia , Conformação de Ácido Nucleico , Pirróis/química , Estreptozocina/farmacologiaRESUMO
Polymers for recovery/removal of the antimicrobial agent oxytetracycline (OTC) from aqueous media were developed with use of computational design and molecular imprinting. 2-Hydroxyethyl methacrylate, 2-acrylamide-2-methylpropane sulfonic acid (AMPS), and mixtures of the two were chosen according to their predicted affinity for OTC and evaluated as functional monomers in molecularly imprinted polymers and nonimprinted polymers. Two levels of AMPS were tested. After bulk polymerization, the polymers were crushed into particles (200-1000 µm). Pressurized liquid extraction was implemented for template removal with a low amount of methanol (less than 20 mL in each extraction) and a few extractions (12-18 for each polymer) in a short period (20 min per extraction). Particle size distribution, microporous structure, and capacity to rebind OTC from aqueous media were evaluated. Adsorption isotherms obtained from OTC solutions (30-110 mg L(-1)) revealed that the polymers prepared with AMPS had the highest affinity for OTC. The uptake capacity depended on the ionic strength as follows: purified water > saline solution (0.9 % NaCl) > seawater (3.5 % NaCl). Polymer particles containing AMPS as a functional monomer showed a remarkable ability to clean water contaminated with OTC. The usefulness of the stationary phase developed for molecularly imprinted solid-phase extraction was also demonstrated. Graphical Abstract Selection of functional monomers by molecular modeling renders polymer networks suitable for removal of pollutants from contaminated aqueous environments, under either dynamic or static conditions.
Assuntos
Antibacterianos/isolamento & purificação , Impressão Molecular/métodos , Oxitetraciclina/isolamento & purificação , Extração em Fase Sólida/métodos , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água/métodos , Acrilamidas/química , Adsorção , Alcanossulfonatos/química , Água Subterrânea/análise , Metacrilatos/química , Modelos Moleculares , PolimerizaçãoRESUMO
A novel core-shell DNA self-assembly catalyzed by thiol-disulfide exchange reactions was proposed, which could realize GSH-initiated hybridization chain reaction (HCR) for signal amplification and molecules gathering. Significantly, these self-assembled products via electrostatic interaction could accumulate into prominent and clustered fluorescence-bright spots in single cancer cells for reduced glutathione monitoring, which will effectively drive cell monitoring into a new era.