Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.872
Filtrar
1.
Proc Natl Acad Sci U S A ; 117(24): 13480-13489, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32487732

RESUMO

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Substâncias Macromoleculares/química , Modelos Químicos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Imagem Individual de Molécula , Viscosidade
2.
Life Sci ; 254: 117752, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387412

RESUMO

AIMS: To design and evaluate novel mono-PEGylated dimeric GLP-1 conjugate with enhanced GLP-1 receptor activation and prolonged anti-diabetes efficacies. MAIN METHODS: All these novel GLP-1 conjugates were produced by using solid-phase synthesis method and further specific cysteine-maleimide modification. In vitro GLP-1R activation assay was performed in CHO cells stably expressing human GLP-1 receptor. The binding affinity for human serum albumin (HSA) in vitro was also conducted using surface plasmon resonance measurement. Subsequently, selected GLP-1 conjugate was subjected to evaluate the acute and chronic efficacies in vivo. KEY FINDINGS: Four novel glucagon-like peptide-1 (GLP-1) conjugates, termed DIG-1 to DIG-4, were designed and prepared with high purity. Moreover, DIG-1(PEG-5 kDa) and DIG-2 (PEG-10 kDa) exerted ~3-fold and ~2-fold higher potencies of GLP-1R activation than native GLP-1, respectively, and both obviously higher than the DIG-3 (PEG-10 kDa) and DIG-4 (PEG-30 kDa). Then DIG-2 exhibited better in vivo glucose-stabilizing and insulinotropic efficacies than DIG-1 by using multiple oral glucose tests (OGTTs) in SD rats. Furthermore, prolonged glucose-lowering ability of DIG-2 exhibited in hypoglycemic duration test and multiple OGTTs in diabetic db/db mice. Pharmacokinetic data of DIG-2 in cynomolgus monkeys revealed a half-life of ~97.2 h and ~120.4 h after a single subcutaneous (s.c.) administration at doses of 100 and 150 nmol/kg, respectively. Chronic treatment of DIG-2 in db/db mice for consecutive 8-week significantly ameliorate the diabetic symptoms including deteriorative % hemoglobin A1C (HbA1C), glucose tolerance and pancreatic function. SIGNIFICANCE: DIG-2, as a novel mono-PEGylated dimeric GLP-1 conjugate, holds enhanced receptor activation and prolonged anti-diabetes efficacies.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipoglicemiantes/uso terapêutico , Polietilenoglicóis/metabolismo , Animais , Células CHO , Cricetulus , Dimerização , Humanos , Ratos
3.
AAPS PharmSciTech ; 21(4): 124, 2020 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-32342227

RESUMO

To achieve improved drug delivery efficiency to hepatocellular carcinoma (HCC), biodegradable poly (ethylene glycol)-poly (lactic-co-glycolic acid) (PEG-PLGA) nanoparticles (NP), surface-modified with SP94 peptide, were designed for the efficient delivery of cryptotanshinone to the tumor for the treatment of HCC. Cryptotanshinone NP and SP94-NP were prepared by using nanoprecipitation. The physicochemical and pharmaceutical properties of the NP and SP94-NP were characterized, and the release kinetics suggested that both NP and SP94-NP provided continuous, slow release of cryptotanshinone for 48 h. The in vitro cellular experiment demonstrated that SP94-NP significantly enhanced the cellular uptake of cryptotanshinone and induced high cytotoxicity and cellular apoptosis of hepatocellular carcinoma (HepG2) cells. The in vivo detecting results of targeting effect using the Cy5.5 probe evidenced that SP94-NP showed an accumulation in tumor more efficiently than that of unconjugated ones. Meanwhile, SP94-NP exhibited the smallest tumor size than other groups and showed no toxicity to body. The results of this study provide a promising nanoplatform for the targeting of HCC.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fenantrenos/administração & dosagem , Poliésteres/administração & dosagem , Polietilenoglicóis/administração & dosagem , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/síntese química , Medicamentos de Ervas Chinesas/metabolismo , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Fenantrenos/síntese química , Fenantrenos/metabolismo , Poliésteres/síntese química , Poliésteres/metabolismo , Polietilenoglicóis/síntese química , Polietilenoglicóis/metabolismo
4.
PLoS One ; 15(3): e0224002, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32191706

RESUMO

Reproducibility of results is essential for a well-designed and conducted experiment. Several reasons may originate failure in reproducing data, such as selective reporting, low statistical power, or poor analysis. In this study, we used PEG6000 samples from different distributors and tested their capability inducing spheroid formation upon surface coating. MALDI-MS, NMR, FTIR, and Triple SEC analysis of the different PEG60000s showed nearly identical physicochemical properties different, with only minor differences in mass and hydrodynamic radius, and AFM analysis showed no significant differences in the surface coatings obtained with the available PEG6000s. Despite these similarities, just one showed a highly reproducible formation of spheroids with different cell lines, such as HT-29, HeLa, Caco2, and PANC-1. Using the peculiar PEG6000 sample and a reference PEG6000 chosen amongst the others as control, we tested the effect of the cell/PEG interaction by incubating cells in the PEG solution prior to cell plating. These experiments indicate that the spheroid formation is due to direct interaction of the polymer with the cells rather than by interaction of cells with the coated surfaces. The experiments point out that for biological entities, such as cells or tissues, even very small differences in impurities or minimal variations in the starting product can have a very strong impact on the reproducibility of data.


Assuntos
Reprodutibilidade dos Testes , Esferoides Celulares/metabolismo , Células CACO-2 , Varredura Diferencial de Calorimetria , Técnicas de Cultura de Células , Cromatografia em Gel , Células HT29 , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
5.
J Nanobiotechnology ; 18(1): 45, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32169073

RESUMO

BACKGROUND: To effectively applied nanomaterials (NMs) in medicine, one of the top priorities is to address a better understanding of the possible sub-organ transfer, clearance routes, and potential toxicity of the NMs in the liver and kidney. RESULTS: Here we explored how the surface chemistry of polyethylene glycol (PEG), chitosan (CS), and polyethylenimine (PEI) capped gold nanoparticles (GNPs) governs their sub-organ biodistribution, transfer, and clearance profiles in the liver and kidney after intravenous injection in mice. The PEG-GNPs maintained dispersion properties in vivo, facilitating passage through the liver sinusoidal endothelium and Disse space, and were captured by hepatocytes and eliminated via the hepatobiliary route. While, the agglomeration/aggregation of CS-GNPs and PEI-GNPs in hepatic Kupffer and endothelial cells led to their long-term accumulation, impeding their elimination. The gene microarray analysis shows that the accumulation of CS-GNPs and PEI-GNPs in the liver induced obvious down-regulation of Cyp4a or Cyp2b related genes, suggesting CS-GNP and PEI-GNP treatment impacted metabolic processes, while the PEI-GNP treatment is related with immune responses. CONCLUSIONS: This study demonstrates that manipulation of nanoparticle surface chemistry can help NPs selectively access distinct cell types and elimination pathways, which help to clinical potential of non-biodegradable NPs.


Assuntos
Ouro/metabolismo , Ouro/toxicidade , Rim/metabolismo , Fígado/metabolismo , Nanopartículas Metálicas/toxicidade , Animais , Quitosana/metabolismo , Citosol , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Ouro/sangue , Rim/patologia , Cinética , Fígado/patologia , Masculino , Nanopartículas Metálicas/química , Camundongos , Camundongos Endogâmicos ICR , Tamanho da Partícula , Polietilenoglicóis/metabolismo , Polietilenoimina/metabolismo , Ratos , Ratos Wistar , Distribuição Tecidual , Transcriptoma
6.
Biochemistry ; 59(7): 831-835, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32022543

RESUMO

Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.


Assuntos
Adenina/análogos & derivados , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Pirrolidinas/metabolismo , Adenina/química , Adenina/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Domínio Catalítico , Inibidores Enzimáticos/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ligação Proteica , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/química , Pirrolidinas/química
7.
Chemistry ; 26(11): 2470-2477, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-31912555

RESUMO

Multidrug resistance (MDR) is regarded as a main obstacle for effective chemotherapy, and P-glycoprotein (P-gp)-mediated drug efflux has been demonstrated to be the key factor responsible for MDR. In this study, a novel pH-responsive hybrid drug delivery system was developed by conjugating d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), a kind of P-gp inhibitor, on the surface of laponite nanodisks to overcome MDR. The prepared LM-TPGS display excellent colloidal stability, a high encapsulation efficiency of doxorubicin (DOX), and a pH-responsive drug release profile. In vitro experiments verified that LM-TPGS/DOX could exhibit significantly enhanced therapeutic efficacy in treating DOX-resistant breast cancer cells (MCF-7/ADR) through inhibiting the activity of P-gp-mediated drug efflux and effectively accumulating DOX within cancer cells. In vivo results revealed that LM-TPGS/DOX outstandingly suppressed MCF-7/ADR tumors with low side effects. Therefore, the high drug payload, enhanced inhibition efficacy to drug-resistant cells, and low side effects make the LM-TPGS/DOX a promising nanoplatform to reverse MDR for effective chemotherapy.


Assuntos
Antibióticos Antineoplásicos/química , Doxorrubicina/química , Nanocápsulas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Composição de Medicamentos/métodos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Vitamina E/química , Vitamina E/metabolismo
8.
Chem Commun (Camb) ; 56(7): 1085-1088, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31894779

RESUMO

We report an elastase-responsive, H2S-releasing hydrogel prepared by covalently crosslinking a mixture of carboxymethylcellulose and poly(ethylene glycol) with an elastase-degradable peptide functionalized with an H2S-releasing S-aroylthiooxime (SATO) unit. Addition of elastase triggered a gel-to-sol transition, which exposed SATOs, leading to more and longer H2S release compared to untriggered gels.


Assuntos
Carboximetilcelulose Sódica/farmacologia , Hidrogéis/farmacologia , Sulfeto de Hidrogênio/metabolismo , Elastase de Leucócito/metabolismo , Polietilenoglicóis/farmacologia , Animais , Carboximetilcelulose Sódica/síntese química , Carboximetilcelulose Sódica/metabolismo , Linhagem Celular , Doxorrubicina/toxicidade , Humanos , Hidrogéis/síntese química , Hidrogéis/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Oximas/síntese química , Oximas/metabolismo , Oximas/farmacologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/metabolismo , Substâncias Protetoras/síntese química , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Ratos
9.
AAPS PharmSciTech ; 21(3): 78, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31970547

RESUMO

Protein drugs were considered to be the first choice to treat many human diseases, but their clinical application was usually limited by their short half-life and lack of validated targeted therapy. Here, a series of folate-functionalized poly(ethylene glycol)-b-(poly(2-aminoethyl-L-glutamate)-g-poly(L-glutamic acid))s (FA-PEG-b-(PELG-g-PLGA)s) were designed as tumor-targeted carriers for cationic protein delivery. Compared with traditional copolymers consisting of PEG and linear charged hydrophilic blocks, FA-PEG-b-(PELG-g-PLGA) with brush-like polyelectrolyte segments were beneficial to improving their electrostatic interactions with loading protein molecules, thus increasing drug-loading stability and protecting encapsulated proteins from degradation. The designed polymer brushes could efficiently encapsulate cytochrome C (CytC), a cationic model protein, to form polyion complex (PIC) micelles with an average particle size of approximately 200 nm. An in vitro drug release study showed that the drug-loading stability of the formed PIC micelles was largely improved. The functionalization of the block copolymer carriers with a targeting folate group enhanced the tumor cell growth inhibition and total apoptotic rates induced by CytC. Our results shed light on the unique advantages of brush-like polymer carriers in delivering cationic proteins, and the poly(L-glutamic acid)-based linear-brush diblock copolymers could be applied as a versatile delivery platform for molecular targeting in cancer therapy.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Ácido Glutâmico/síntese química , Poliésteres/síntese química , Polietilenoglicóis/síntese química , Proteínas/síntese química , Animais , Cátions , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Liberação Controlada de Fármacos , Ácido Glutâmico/administração & dosagem , Ácido Glutâmico/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Células NIH 3T3 , Tamanho da Partícula , Poliésteres/administração & dosagem , Poliésteres/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Polímeros/administração & dosagem , Polímeros/síntese química , Polímeros/metabolismo , Proteínas/administração & dosagem , Proteínas/metabolismo
10.
Biochim Biophys Acta Gen Subj ; 1864(4): 129541, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31987956

RESUMO

BACKGROUND: BAX activation is a crucial step for commitment to apoptosis. Several activators, such as BimBH3-based therapeutic peptides and cleaved Bid (cBid) protein, can trigger BAX-mediated apoptosis, but it is unclear whether they proceed through the same pathway. METHODS: Here we utilize PEGylation-based approach, which is shown to efficiently shield individual binding grooves in BAX from activators, to investigate and reveal that the activators take different routes to induce BAX-mediated apoptosis. Various spectroscopic/biochemical tools, including electron spin resonance, circular dichroism, fluorescence recovery after photobleaching, and label-transfer assay, were employed to reveal details in the processes. RESULTS: We observe a key mutant BAX 164-PEG that acts differently in response to cBid and BimBH3 stimuli. While BimBH3 directly interacts with the trigger groove (TG) to induce the conformational changes in BAX that includes the release of α9 from the canonical groove (CG) and oligomerization, cBid engages with CG and works with mitochondrial lipids to fully activate BAX. CONCLUSION: PEGylation-based approach is proven useful to shield individual binding grooves of BAX from apoptotic stimuli. Groove engagement in CG of BAX is required for a full cBid-induced BAX activation. This study has identified differences in the pathways involved during the initiation of BAX activation by full-length cBid protein versus synthetic BimBH3-based peptides. GENERAL SIGNIFICANCE: Our finding is potentially valuable for therapeutic application as the pore-forming activity of 164-PEG is independent from the cBid-mediated apoptotic pathways, but can be administrated by the synthetic short peptides.


Assuntos
Polietilenoglicóis/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Camundongos , Polietilenoglicóis/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética
11.
Biointerphases ; 14(6): 061005, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896261

RESUMO

Extracellular matrix provides critical signaling context to resident cells through mechanical and bioactive properties. To realize the potential of tissue engineering and regenerative medicine, biomaterials should allow for the independent control of these features. This study investigates a hydrogel system based on thiol-modified hyaluronic acid (HA-S) and polyethylene glycol diacrylate (PEGDA). The mechanical properties of HAS-PEGDA are dictated by two cytocompatible crosslinking reactions that occur at distinct time points: a rapid, Michael-type nucleophilic addition reaction between HA-thiols and PEG-acrylates and a prolonged maturation of disulfide crosslinks from remaining thiols. It is hypothesized that these reactions would enable the independent tuning of the mechanical and bioactive features of HAS-PEGDA. Rheological studies confirmed that initial gelation reached completion by 1 day, at which point the shear modulus was proportional to the concentration of PEGDA. Over time, the shear modulus evolved dramatically, and final stiffness depended on the availability of HA-thiols. The addition of PEG-monoacrylate (PEGMA) after the initial gelation occupied a percentage of remaining thiols to prevent disulfide crosslinking, decreasing the steady-state stiffness in a dose-dependent manner. A fraction of the PEGMA was then replaced with acrylated peptide ligands to introduce specific bioactivity to the otherwise non-cell-adhesive network. The degree of latent stiffening was controlled by the total amount of peptide-PEGMA, while adhesivity was tuned with the balance of bioactive and inactive peptides. The functional effects of the tunable mechanical and bioadhesive ligand properties were confirmed with assays of cell adhesion and morphology.


Assuntos
Fenômenos Biomecânicos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hidrogéis/síntese química , Hidrogéis/metabolismo , Acrilatos/química , Acrilatos/metabolismo , Animais , Adesão Celular , Forma Celular , Células Cultivadas , Elasticidade , Fibroblastos/efeitos dos fármacos , Hidrogéis/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ratos , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Viscosidade
12.
Talanta ; 208: 120358, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816795

RESUMO

The PEGylated liposomal nanoparticle has been widely used as a carrier in drug delivery system. To become biologically active, the encapsulated drug must be released from the nanoparticle vehicle. However, due to limitations of current bioanalytical methods, the characterization of this release process has been restricted to determination of total drug in tissues and tumor. As a result, the fate of liposomal nanoparticles including their uptake into target tissue has not been fully characterized. In this study, we developed a novel two-step solid phase extraction on two separated columns procedure to separate liposomes from tissues and tumors without liposomal leakage. This allowed us to determine encapsulated drug, total drug and, by difference, released drug and compare the release and uptake profiles of PEGylated liposomal doxorubicin in tissues and tumor of tumor-bearing mice with corresponding profiles for free doxorubicin. The liposomal nanoparticles released doxorubicin into tumor efficiently and, compared with administration of free drug, increased doxorubicin uptake into tumor by 1.8-fold. It also decreased doxorubicin uptake into heart (0.78-fold lower) with the potential to reduce doxorubicin cardiotoxicity. Drug release reached constant levels in tissues and tumor after 12 h with released doxorubicin concentration remaining at 70-80% of total doxorubicin concentration and in tumor at 86% of total drug concentration. The assay also included determination of the main doxorubicin metabolites. Determination of the metabolites showed that liposomal entrapment delays and decreases the metabolism of doxorubicin but does not alter the metabolic pathway. These results provide a clear and comprehensive picture of the biodistribution of doxorubicin administered in liposomal nanoparticles which may assist in the rational design of other liposomal nanoparticles.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas/administração & dosagem , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/metabolismo , Apoptose , Transporte Biológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Doxorrubicina/administração & dosagem , Doxorrubicina/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Immunol Methods ; 474: 112669, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31614128

RESUMO

Detection of anti-drug antibodies is a critical step in the development of large molecule biopharmaceuticals. In the case of multicomponent/multifunctional molecules, such as fusion proteins and protein conjugates such as covalent polyethylene glycol (PEG)~protein conjugates, it is useful to further characterize anti-drug antibody (ADA) binding to key domains of the drug. The detection of anti-PEG antibodies poses special challenges that if overlooked can result in underreporting antibody responses. Here we describe the development and characterization of a novel ELISA to detect anti-PEG antibodies that provides a more complete interpretation of anti-PEG than other published methods. Being specific to the PEG moiety alone, this method is intended to detect anti-PEG antibodies independent of the protein to which PEG is conjugated. Based upon early indications that our assay could detect anti-PEG antibodies at a surprisingly high frequency in the general population, our emphasis throughout method development and validation was to ensure that non-specific signals and unintended interactions were not falsely contributing to detection of anti-PEG antibodies. Techniques, including orthogonal methods used to ensure that this ELISA detected antibodies specific to PEG included competition, immunodepletion, immunoprecipitation/western blot and an Octet kinetic binding analysis. The validated ELISA can detect 100 ng/mL of an anti-PEG IgG positive control and 800 ng/mL of an anti-PEG IgM positive control in the presence of 7.5 µg/mL of the PEGylated therapeutic (MW 64 kDa). The intra-assay percent co-efficient of variation (CV) and inter-assay CV of the low positive control samples in the screening method were 4.1 to 7.2% and 16.7 to 17.7%, respectively. Additional assay performance parameters that were validated are also described. When the validated assay was applied to a population of 200 healthy blood donors with no known exposure to biopharmaceutical PEG conjugates it indicated a pre-existing anti-PEG antibody prevalence of 97.5%. We suggest this surprising result is a consequence of exposure to PEG additives in everyday products, such as cosmetics, processed foods and over-the-counter (OTC) pharmaceuticals.


Assuntos
Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Polietilenoglicóis , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Humanos , Cinética , Polietilenoglicóis/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Tensoativos/química
14.
Eur J Pharm Biopharm ; 145: 27-34, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31629787

RESUMO

Exosomes are gaining increasing attention as drug delivery vehicles due to their low toxicity and ability to functionally transfer biological cargos between cells. However, the therapeutic applicability of exosomes is partially hampered by a lack of cell-type specificity. In this study, therefore, we investigated the impact of cell-type tropism on the in vivo systemic delivery of exosomes to tumor tissues. Exosomes derived from murine colorectal cancer cells (C26) (C26-Exos) and murine melanoma cells (B16BL6) (B16BL6-Exos) were collected. In vitro cellular uptake of either autologous (C26) or allogeneic (B16BL6) exosomes by C26 tumor cells was determined. In vivo tumor accumulation of each type of exosomes in mice bearing C26 tumors was monitored with an in vivo imaging system (IVIS). In in vitro studies, autologous C26-Exos were more efficiently taken up by C26 cancer cells, compared to allogeneic B16BL6-Exos. For in vivo studies, exosomes were modified with surface polyethylene glycol (PEG) to improve their circulation lifetimes. Although both types of PEGylated exosomes accumulated in C26-tumor tissue, autologous exosomes were preferentially accumulated within C26-tumor tissue compared to allogeneic exosomes. The increased tumor accumulation of autologous PEGylated exosomes was accompanied by the preferential uptake of exosomes by not only C26-tumor cells but also tumor-associated immune cells. This study implies that cancer cell-type tropism is an important factor in the achievement of tumor cell targeting with cancer cell-derived exosomes.


Assuntos
Neoplasias Colorretais/metabolismo , Exossomos/metabolismo , Melanoma/metabolismo , Tropismo/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/metabolismo
15.
Anal Bioanal Chem ; 411(27): 7087-7094, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471684

RESUMO

Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.


Assuntos
Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/análogos & derivados , Esferoides Celulares/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Células HCT116 , Humanos , Espectrometria de Massas , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Tripsina/metabolismo
16.
Colloids Surf B Biointerfaces ; 183: 110491, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518956

RESUMO

Recently, molybdenum disulfide functionalized with poly-ethylene glycol (PEGylated MoS2) has been widely used as a new drug delivery vehicle in biomedical field. However, the weak antibacterial activity of PEGylated MoS2 limits its application as an antibacterial agent. In this work, a novel silkworm-like conjugate of nisin loaded PEGylated MoS2 (nisin@PEGylated MoS2) was developed for antibacterial application. The morphology and structure of PEGylated MoS2 were strongly dependent on the Mo/S molar ratio of precursors during the solvothermal process. The silkworm-like skeleton was well kept after loading with nisin. A high level of reactive oxygen species (ROS) induced by the conjugate was an important cause of bacteria death. Due to the different structure of cell membranes, the sharp edges could more easily puncture into Escherichia coli (E. coli) as compared with Staphylococcus aureus (S. aureus) and produced more intracellular ROS, which improved the antibacterial activity of nisin against E. coli. As a result, nisin@PEGylated MoS2 displayed the antibacterial activity against both gram-positive and gram-negative bacteria. Furthermore, the toxicity of the conjugate was very low. Therefore, the target conjugate of nisin@PEGylated MoS2 may have great potential application as an antibacterial agent.


Assuntos
Antibacterianos/farmacologia , Dissulfetos/química , Proteínas de Insetos/química , Molibdênio/química , Nisina/química , Polietilenoglicóis/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Bombyx/metabolismo , Dissulfetos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Insetos/metabolismo , Molibdênio/metabolismo , Nisina/metabolismo , Polietilenoglicóis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
17.
Soft Matter ; 15(35): 7071-7079, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31441486

RESUMO

Motile bacteria are often found in complex, polymer-rich environments in which microbes can aggregate via polymer-induced depletion forces. Bacterial aggregation has many biological implications; it can promote biofilm formation, upregulate virulence factors, and lead to quorum sensing. The steady state aggregation behavior of motile bacteria in polymer solutions has been well studied and shows that stronger depletion forces are required to aggregate motile bacteria as compared with their nonmotile analogs. However, no one has studied whether these same trends hold at the initial stages of aggregation. We use experiments and numerical calculations to investigate the polymer-induced depletion aggregation of motile Escherichia coli in polyethylene glycol solutions on short experimental timescales (∼10 min). Our work reveals that in the semi-dilute polymer concentration regime and at short timescales, in contrast to what is found at steady state, bacterial motility actually enhances aggregate formation by increasing the collision rate in viscous environments. These unexpected findings have implications for developing models of active matter, and for understanding bacterial aggregation in dynamic, biological environments, where the system may never reach steady state.


Assuntos
Movimento Celular , Quimiotaxia , Escherichia coli/fisiologia , Polietilenoglicóis/metabolismo , Polímeros/metabolismo , Percepção de Quorum , Biofilmes , Polietilenoglicóis/química , Polímeros/química
18.
Colloids Surf B Biointerfaces ; 183: 110412, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31398620

RESUMO

miRNAs such as miR-148b play crucial regulatory role in tumor metastasis, but their applications are limited because they are easy to degrade in serum conditions and lack targeting ability. Herein, CC9-PEG-SSBPEI was synthesized and used as nano-carrier for miR-148b. DLS and gel retardation analyses indicated that CC9-PEG-SSBPEI could combine with miR-148b by charge interaction and formed into nanoparticles with the size changed from 811.6 nm to 146.4 nm. CC9-PEG-SSBPEI could protect miR-148b from RNase A degradation and showed a reduction sensitive release of miR-148b. FACS analysis and CLSM images displayed that the conjugated CC9 peptide improved the accumulation and penetration of the nanoparticles in HuH-7 liver cancer cells through binding with the target of miR-148b neuropilin-1(NRP-1) on the cell surface. The raised level of miR-148b in turn inhibited the expression of NRP-1 and suppressed the migration of HuH-7 liver cancer cells. Moreover, hemolysis and cytotoxicity assay demonstrated that the nanoparticles had good hemo- and cyto- compatibility. Hence, CC9-PEG-SSBPEI/miR-148b nanoparticles had the potential for targeting delivery of miR-148b and anti-metastasis of hepatocellular carcinoma (HCC) cells.


Assuntos
Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Técnicas de Transferência de Genes , MicroRNAs/genética , Nanopartículas/química , Neuropilina-1/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Eritrócitos/citologia , Hemólise , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oxirredução , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoimina/química , Polietilenoimina/metabolismo , Ribonuclease Pancreático/química
19.
Colloids Surf B Biointerfaces ; 183: 110413, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31401461

RESUMO

A novel bioinspired nanoplatform capable of fast endocytosis, lysosomal pH-triggered drug release, and reduced drug efflux based on PBA-PEG-b-P(Glu-co-GluDA) copolymer was developed in this study. The synthesized copolymer could facilitate doxorubicin encapsulation with relatively high drug-loading content and efficiency. Inspired by mussel byssal threads, a core crosslinking strategy based on the coordination between catechol and ferric ions was introduced to improve the stability of nanomicelles and realize lysosomal pH-controlled drug release. This nanoplatform could maintain integrity even after being dissolved in a good solvent, demonstrating its the potential to withstand infinite dilution of plasma after intravenous injection. Moreover, this nanoplatform demonstrated lysosomal pH-triggered drug release, and the cumulative release amount of doxorubicin under a simulated lysosomal condition was 13 times higher than that under a simulated plasma condition. Moreover, as a result of the high binding capacity between phenylboronic acid (PBA) and sialic acid on the surface of human hepatoma cell line (HepG2), the fast and enhanced endocytosis in addition to lysosomal pH-triggered release property and significantly low efflux, this nanoplatform exhibits improved delivery efficiency of doxorubicin into the nucleus and notably outstanding antiproliferative effects compared with doxorubicin. Furthermore, the PBA modification remarkably increased the mean fluorescence intensity of this nanoplatform endocytosed by HepG2 cells to twice that of doxorubicin after one hour of incubation. The nanoplatform exhibited an inhibition rate of 70% against tumor growth. Thus, this novel nanoplatform based on PBA-PEG-b-P(Glu-co-GluDA) copolymer displayed multifunctionality and exhibited great potential as an intelligent nanoplatform for antitumor drug delivery.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Núcleo Celular/efeitos dos fármacos , Preparações de Ação Retardada , Doxorrubicina/farmacologia , Lisossomos/efeitos dos fármacos , Nanopartículas/química , Antibióticos Antineoplásicos/metabolismo , Materiais Biomiméticos/química , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Catecóis/química , Catecóis/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Composição de Medicamentos/métodos , Endocitose , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Ferro/química , Ferro/metabolismo , Lisossomos/metabolismo , Micelas , Nanopartículas/ultraestrutura , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo
20.
Drug Dev Ind Pharm ; 45(10): 1707-1715, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418304

RESUMO

Numerous normal and tumors cells are well-known to express the somatostatin receptors (SSTRs) on their surface which makes the receptor be useful for tumor scintigraphy. Thus, the identification of SSTRs is beneficial, especially SSTR2. The somatostatin analog, Octreotide (OCT), was chosen as a ligand, as it is known to selectively bind to SSTR2. Moreover, polyethylene glycol (PEG), 8armPEG, was used as a branched PEG to provide a low nonspecific cell binding and easily chemical modification. OCT and fluorescein (Flu) were conjugated to branched PEG using a water-soluble carbodiimide (EDC) and N-hydroxy succinimide (NHS) so as to activate its carboxylic acid group. 8armPEG-tagged Flu and OCT was characterized by gel permeation chromatography (GPC) to proof the conjugation of OCT to 8armPEG. Finally, cellular uptake was studied using pancreatic cancer cells with well-expressed somatostatin receptors using a confocal laser scanning microscope (CLMS) and fluorescence activated cell sorting (FACS). GPC showed increases in molecular mass since it showed a difference in elution time of 8armPEG itself and 8armPEG labeled with Flu. CLMS and FACS showed high binding with the positive SSTR2 cells expression and showed negative results with negative expressing SSTR2. These bindings were decreased when the receptors were occupied with free OCT which confirms the specific binding to SSTR2. Therefore, we formulated a novel model to easily identify SSTR2 and other receptors which serves as a promising platform for identification of tumor cells overexpressing the SSTR2, which would be a hopeful target for cancer therapy and tumor scintigraphy.


Assuntos
Octreotida/metabolismo , Polietilenoglicóis/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Células HCT116 , Humanos , Células MCF-7 , Neoplasias Pancreáticas/metabolismo , Cintilografia/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA