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1.
Nat Commun ; 12(1): 5552, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34548486

RESUMO

Sepsis is a life-threatening condition caused by the extreme release of inflammatory mediators into the blood in response to infection (e.g., bacterial infection, COVID-19), resulting in the dysfunction of multiple organs. Currently, there is no direct treatment for sepsis. Here we report an abiotic hydrogel nanoparticle (HNP) as a potential therapeutic agent for late-stage sepsis. The HNP captures and neutralizes all variants of histones, a major inflammatory mediator released during sepsis. The highly optimized HNP has high capacity and long-term circulation capability for the selective sequestration and neutralization of histones. Intravenous injection of the HNP protects mice against a lethal dose of histones through the inhibition of platelet aggregation and migration into the lungs. In vivo administration in murine sepsis model mice results in near complete survival. These results establish the potential for synthetic, nonbiological polymer hydrogel sequestrants as a new intervention strategy for sepsis therapy and adds to our understanding of the importance of histones to this condition.


Assuntos
Hidrogéis/uso terapêutico , Nanopartículas/uso terapêutico , Sepse/tratamento farmacológico , Animais , Plaquetas/efeitos dos fármacos , Adesão Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Histonas/antagonistas & inibidores , Histonas/metabolismo , Histonas/toxicidade , Hidrogéis/química , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Nanopartículas/química , Nanopartículas/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Ligação Proteica , Sepse/mortalidade , Taxa de Sobrevida
2.
Artigo em Inglês | MEDLINE | ID: mdl-32361632

RESUMO

Edoxaban is mainly enzymatically converted to a 4-carboxylic acid form (4CA-EDX) and an N-desmethyl form (ND-EDX) in humans. This study aimed to establish a simple liquid chromatography-tandem mass spectrometry method using core-shell octadecyl silica (ODS) microparticles for the simultaneous quantitation of edoxaban and its two major metabolites in human plasma. Analytes extracted from plasma specimens by a one-step deproteinization were separated using a 2.6-µm core-shell ODS microparticulate column and linear acetonitrile-ammonium acetate gradient elution at a flow rate of 0.25 mL/min with a run time of 7 min. The mass spectrometer was operated in the positive ion multiple reaction monitoring mode. Plasma samples collected from 20 patients with atrial fibrillation were analyzed by the present method. The chromatograms of drug-free human plasma had no interfering peaks. The calibration curves of edoxaban, 4CA-EDX, and ND-EDX were linear over the concentration ranges of 1.25-160, 0.47-60, and 0.12-15 ng/mL, respectively. Their pretreatment recoveries and matrix factors were 88.7-109.0% and 87.0-101.6%, respectively. The intra- and inter-day accuracy and imprecision values were 85.9-112.8% and within 13.3%, respectively. The plasma concentrations of edoxaban, 4CA-EDX, and ND-EDX in the patients had ranges of 17.8-102, 1.67-25.7, and 0.685-5.34 ng/mL, respectively. All the analytes were measurable within their calibration curves. In conclusion, this validated method for the simultaneous determination of edoxaban and its major metabolites was successfully applied to plasma specimens obtained from patients with atrial fibrillation.


Assuntos
Piridinas/sangue , Piridinas/farmacocinética , Espectrometria de Massas em Tandem/métodos , Tiazóis/sangue , Tiazóis/farmacocinética , Técnicas Biossensoriais , Coleta de Amostras Sanguíneas , Cromatografia Líquida de Alta Pressão , Humanos , Limite de Detecção , Metabolômica , Microesferas , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Dióxido de Silício/química
3.
Biomacromolecules ; 20(10): 3648-3657, 2019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31518109

RESUMO

The affinity of a synthetic polymer nanoparticle (NP) to a target biomacromolecule is determined by the association and dissociation rate constants (kon, koff) of the interaction. The individual rates and their sensitivity to local environmental influences are important factors for the on-demand capture and release a target biomacromolecule. Positively charged NPs for small interfering RNA (siRNA) delivery is a case in point. The knockdown efficacy of siRNA can be strongly influenced by the binding kinetics to the NP. Here, we show that kon and koff of siRNA to NPs can be individually engineered by tuning the chemical structure and composition of the NP. N-Isopropylacrylamide-based NPs functionalized with hydrophobic and amine monomers were used. koff decreased by increasing the amount of amine groups in the NP, whereas kon did not change. Importantly, NPs showing a low koff at pH 5.5 together with a high koff at pH 7.4 showed high knockdown efficiency when NP/siRNA complexes were packaged in lipid nanoparticles. These results provide direct evidence for the premise that the efficacy of an siRNA delivery vector is linked with the strong affinity to the siRNA in the endosome and low affinity in the cytoplasm.


Assuntos
Técnicas de Transferência de Genes , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Acrilamidas/química , Animais , Linhagem Celular Tumoral , Citoplasma/metabolismo , Endossomos/metabolismo , Técnicas de Silenciamento de Genes/métodos , Camundongos , RNA Interferente Pequeno/genética , Polímeros Responsivos a Estímulos/química
4.
J Control Release ; 295: 13-20, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30578808

RESUMO

Protein affinity reagents (PARs), frequently antibodies, are essential tools for basic research, diagnostics, separations and for clinical applications. However, there is growing concern about the reproducibility, quality and cost of recombinant and animal-derived antibodies. This has prompted the development of alternatives that could offer economic, and time-saving advantages without the use of living organisms. Synthetic copolymer nanoparticles (NPs), engineered with affinity for specific protein targets, are potential alternatives to PARs. Although there are now a number of examples of abiotic protein affinity reagents (APARs), most have been evaluated in vitro limiting a realistic assessment of their potential for more demanding, practical in vivo applications. We demonstrate for the first time that an abiotic copolymer hydrogel nanoparticle (NP1) engineered to bind a key signaling protein, vascular endothelial growth factor (VEGF165), functions in vivo to suppress tumor growth by regulating angiogenesis. Lightly cross-linked N-isopropylacrylamide based NPs that incorporate both sulfated N-acetylglucosamine and hydrophobic monomers were optimized by dynamic chemical evolution for VEGF165 affinity. NP1 efficacy in vivo was evaluated by systemic administration to tumor-bearing mice. The study found that NP1 suppresses tumor growth and reduces tumor vasculature density. Combination therapy with doxorubicin resulted in increased doxorubicin concentration in the tumor and dramatic inhibition of tumor growth. NP1 treatment did not show off target anti-coagulant activity. In addition, >97% of injected NPs are rapidly excreted from the body following IV injection. These results establish the use of APARs as inhibitors of protein-protein interactions in vivo and may point the way to their broader use as abiotic, cost effective protein affinity reagents for the treatment of certain cancers and more broadly for regulating signal transduction.


Assuntos
Acrilamidas/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Acrilamidas/administração & dosagem , Acrilamidas/química , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Humanos , Masculino , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
J Control Release ; 268: 335-342, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29061513

RESUMO

Many of macromolecular toxins induce cell death by directly interacting with cells or induction of inflammatory cytokines. Abiotic polymer ligands (PLs) composed of functional monomers are able to bind and neutralize toxins in vivo and are of great interest for efficient antidotes. However, little has been reported about recognition and neutralization of target molecules in the bloodstream because of readily elimination from the bloodstream. Here, we report a rational design of PLs-decorated lipid nanoparticles (PL-NPs) for neutralizing a target toxin in vivo. PL that decorated on the NPs would cooperatively interacts with target biomacromolecules since the lipid molecules in NPs have a high degree of freedom. In the present study, N-isopropylacrylamide based PLs interacting with histones, major mediators of sepsis, were synthesized. Affinity between PL-NPs and histones depends on monomer composition and polymer length. The optimized PL-NP showed little affinity for plasma proteins. The PL-NPs inhibited the toxicity of histones both in vitro and in vivo, suggesting that PLs on the NPs cooperatively bound to histones and neutralized their toxicity. In addition, circulation time of optimized PL was significantly prolonged by the modification onto NPs. These results provide a platform for designing antidote nanoparticles neutralizing toxic biomacromolecules.


Assuntos
Antídotos/administração & dosagem , Histonas/toxicidade , Nanopartículas/administração & dosagem , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Ligantes , Lipídeos/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Polímeros/administração & dosagem
6.
Nat Chem ; 9(7): 715-722, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28644480

RESUMO

Protein affinity reagents are widely used in basic research, diagnostics and separations and for clinical applications, the most common of which are antibodies. However, they often suffer from high cost, and difficulties in their development, production and storage. Here we show that a synthetic polymer nanoparticle (NP) can be engineered to have many of the functions of a protein affinity reagent. Polymer NPs with nM affinity to a key vascular endothelial growth factor (VEGF165) inhibit binding of the signalling protein to its receptor VEGFR-2, preventing receptor phosphorylation and downstream VEGF165-dependent endothelial cell migration and invasion into the extracellular matrix. In addition, the NPs inhibit VEGF-mediated new blood vessel formation in Matrigel plugs in vivo. Importantly, the non-toxic NPs were not found to exhibit off-target activity. These results support the assertion that synthetic polymers offer a new paradigm in the search for abiotic protein affinity reagents by providing many of the functions of their protein counterparts.


Assuntos
Nanopartículas/química , Polímeros/química , Engenharia de Proteínas , Fator A de Crescimento do Endotélio Vascular/química , Proliferação de Células , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
J Control Release ; 228: 1-8, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-26826309

RESUMO

Small interfering RNA (siRNA) has the potential to be a candidate as a cure for intractable diseases. However, an appropriate vector is required for siRNA delivery because of the low transfection efficiency of siRNA without a vector and its easy degradation in vivo. Here, we report a simple, only one step, and efficient method for siRNA encapsulation into a lipidic nanocarrier by freeze-thawing: siRNA was entrapped between the lipid layers of multi-layer liposomes by freeze-thawing of lipoplexes composed of polycation liposomes (PCLs) and siRNA. siRNA-holding capacity to the PCL was increased by repeating freeze-thaw of the lipoplex up to 5cycles. Although siRNA in the conventional lipoplex was degraded after incubation in 90% fetal bovine serum for 72h, siRNA in the frozen and thawed lipoplex was not degraded. Interestingly, we found that the lipoplex formed a "packed multi-layer" structure after the freeze-thawing of "single-layer" PCL and siRNA complex, suggesting that siRNA exists between the lipid layers working as a binder. The frozen and thawed lipoplex showed significantly higher knockdown efficacy compared with the conventional lipoplex. In addition, PEGylated freeze-thawed lipoplexes delivered a higher amount of siRNA to a tumor in vivo compared with the PEGylated conventional ones. These results provide an attractive strategy for "one-step" encapsulation of siRNA into liposomes by freeze-thawing.


Assuntos
Lipossomos/química , Poliaminas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Congelamento , Lipídeos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polieletrólitos , Polietilenoglicóis/química , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Transfecção
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