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1.
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
2.
J Sci Food Agric ; 99(14): 6315-6323, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31260112

RESUMO

BACKGROUND: To increase the low residual activity of levansucrase during long-time processing, an enhancement of its weak thermostability is needed. Here, the effect of metal ions and polyethylene glycol (PEG) on the thermostability of levansucrase from Brenneria sp. EniD312 were studied and evaluated. The residual activity was determined and the protein structure was evaluated by circular dichroism spectrum, fluorescence intensity (FI), and surface hydrophobicity (S0 ). RESULTS: As a result of incubation with 10% (w/v) PEG 4000, the enzyme activity was increased by 1.24-fold. After incubation with 5% PEG 4000 for 6 h, the residual activity at 35 and 45 °C was decreased to 55% and 60% of the initial activity, with an increase of 1.2- and 3.3-fold than the wild-type enzyme. Furthermore, the random coil content of enzyme was decreased from 53% of the wild-type enzyme to 33.9% of the PEG pre-incubated enzyme. Additionally, the FI was maximally increased and the S0 was decreased from 117 to 69. CONCLUSION: All of these results suggested that after incubation with PEG 4000, the secondary and tertiary structure of wild-type enzyme could be greatly maintained and then its thermostability could be increased. This study was the first report on the enhancement of levansucrase thermostability by PEG incubation and might be a good guideline to other researches on levansucrase. © 2019 Society of Chemical Industry.


Assuntos
Proteínas de Bactérias/química , Enterobacteriaceae/enzimologia , Hexosiltransferases/química , Polietilenoglicóis/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Enterobacteriaceae/química , Enterobacteriaceae/genética , Estabilidade Enzimática , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/metabolismo
3.
AAPS PharmSciTech ; 20(5): 218, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31187334

RESUMO

The aim of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for enhancement of the oral bioavailability of isoliquiritigenin (ISL) as well as evaluate its in vivo anti-hyperuricemic effect in rats. The ISL-loaded self-microemulsifying drug delivery system (ISL-SMEDDS) was comprised of ethyl oleate (EO, oil phase), Tween 80 (surfactant), and PEG 400 (co-surfactant). The ISL-SMEDDS exhibited an acceptable narrow size distribution (44.78 ± 0.35 nm), negative zeta potential (- 10.67 ± 0.86 mV), and high encapsulation efficiency (98.17 ± 0.24%). The in vitro release study indicated that the release rates of the formulation were obviously higher in different release media (HCl, pH 1.2; PBS, pH 6.8; double-distilled water, pH 7.0) compared with the ISL solution. The oral bioavailability of the ISL-SMEDDS was enhanced by 4.71 times in comparison with the free ISL solution. More importantly, ISL-SMEDDS significantly reduced uric acid level by inhibiting xanthine oxidase (XOD) activity in the model rats. Collectively, the prepared ISL-SMEDDS proved to be potential carriers for enhancing the solubility and oral bioavailability of ISL, as well as ameliorating its anti-hyperuricemic effect.


Assuntos
Chalconas/administração & dosagem , Chalconas/sangue , Sistemas de Liberação de Medicamentos/métodos , Hiperuricemia/sangue , Hiperuricemia/tratamento farmacológico , Administração Oral , Animais , Disponibilidade Biológica , Emulsões , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/sangue , Masculino , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Ratos , Ratos Sprague-Dawley , Solubilidade , Tensoativos/administração & dosagem , Tensoativos/metabolismo
4.
Carbohydr Polym ; 216: 332-342, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047074

RESUMO

Chitosan has received a lot of attention as a carrier for small interfering RNA (siRNA), due to its capacity for complexation and intracellular release of these molecules. However, one of its limitations is its insolubility at neutral pH and the tendency towards aggregation of its nanoparticles in isotonic ionic strength. In this study, a series of amphipathic chitosans were synthesized by varying the degree of acetylation (DA) from ˜2 to ˜30 mol% and the degree of substitution (DS) from 5 to 25%. by tertiary amino groups (DEAE) The results showed that the adjustment of these parameters decreases the interparticle interactions mediated by hydrogen bonding to obtain nanoparticles with improved colloidal stability. siRNA-containing nanoparticles of 100 to 150 nm with low polydispersities (0.15-0.2) and slightly positive zeta potentials (˜+ 5 mV) were resistant to aggregation at pH 7.4 and ionic strength of 150 mM. This resistance to aggregation is provided by changes on the nanoparticle surface and highlights the importance of more organized self-assembly in providing colloidal stability at physiological conditions. Additionally, the PEGylation of the most promising vectors conferred favorable physicochemical properties to nanoparticles. The chitosans and their nanoparticles exhibited low toxicity and an efficient cell uptake, as probed by confocal microscopy of rhodamine labeled vectors. The results provide a new approach to overcome the limited stability of chitosan nanoparticles at physiological conditions and show the potential of these amphipathic chitosans as siRNA carriers.


Assuntos
Quitosana/análogos & derivados , Portadores de Fármacos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Anidridos Acéticos/química , Acetilação , Animais , Quitosana/síntese química , Quitosana/metabolismo , Quitosana/toxicidade , Dietilaminas/química , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Portadores de Fármacos/toxicidade , Fluorescência , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Camundongos , Nanopartículas/metabolismo , Nanopartículas/toxicidade , Tamanho da Partícula , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoglicóis/toxicidade , Células RAW 264.7 , RNA Interferente Pequeno/química , Rodaminas/química , Tensoativos/síntese química , Tensoativos/metabolismo , Tensoativos/toxicidade
5.
Chem Commun (Camb) ; 55(39): 5540-5546, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31033990

RESUMO

The potential risks associated with two-dimensional (2D) nanomaterials may cause serious concerns about their real applications and impact in biological systems. In addition, the demonstration of biodegradability of these flat nanomaterials is essential in living organisms. Here, we summarise the state-of-the-art in the field of biocompatibility and biodegradability of graphene-related materials (such as 2D materials like MoS2, BN or WS2). The impact of chemical functionalisation on the potential control of the biodegradability profile of these structures is also discussed.


Assuntos
Materiais Biocompatíveis/química , Grafite/química , Animais , Materiais Biocompatíveis/metabolismo , Compostos de Boro/química , Bovinos , Dissulfetos/química , Molibdênio/química , Nanoestruturas/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Soroalbumina Bovina/metabolismo , Tungstênio/química
6.
Mater Sci Eng C Mater Biol Appl ; 100: 141-151, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948048

RESUMO

Macromolecular bioactives, like proteins and peptides, emerged as highly efficient therapeutics. The main limitation for their clinical application is their instability and potential immunogenicity. Thus, controlled delivery systems able protect the proteins prior release are highly on demand. In the present study, we developed hydrophilic thermo-responsive nanogels with tunable volume phase transition temperatures (VPTTs) and suitable features for controlled protein delivery by the use of multifunctional, dendritic polyglycerol (dPG) as macromolecular cross-linker and temperature-sensitive polymers poly(N-isopropylacrylamide) (NIPAM) and poly(N-isopropylacrylmethacrylate) as linear counterpart. We comprehensively studied the impact of the initiator, monomers and cross-linker on the nanogel structure during the synthesis. Careful analysis of the polymerization process revealed importance of balanced reactions kinetics to form particles with diameters in the range 100-200 nm and low polydispersity. We can control the cross-linking density of the nanogels mainly by the dPG feed and its degree of acrylation. In addition, our screenings revealed that the hydrophilic character of dPG enables it to stabilize the growing particles during the polymerization and thereby reduces final particle size. Co-polymerization of NIPAM and NIPMAM allows precise tuning of the VPTT of the nanogels in the desired range of 34-47 °C. Our nanogels showed outstanding high protein encapsulation efficiency and triggered cargo release upon a temperature change. The delivery efficiency of these nanogels was investigated on excised human skin demonstrating efficient dermal penetration of encapsulated proteins dependent on a temperature triggered release mechanism.


Assuntos
Peptídeos/metabolismo , Polietilenoglicóis/química , Polietilenoimina/química , Proteínas/metabolismo , Resinas Acrílicas/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Portadores de Fármacos/química , Glicerol/química , Células HeLa , Humanos , Microscopia de Fluorescência , Peptídeos/química , Polietilenoglicóis/metabolismo , Polietilenoimina/metabolismo , Polímeros/química , Proteínas/química , Temperatura Ambiente
7.
Bioresour Technol ; 283: 67-75, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30901590

RESUMO

A process strategy to aid in optimal enzymatic hydrolysis through the addition of polyethylene glycol (PEG6000) was tested for separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). Pretreated wheat straw at 30% solids (w/w) loading was enzymatically hydrolyzed with 0, 0.5, 1, 1.5, 2 and 2.5% of PEG6000 through SHF and SSF. During SHF, bioethanol concentration of 107.5 g/L (2.5% PEG6000) was achieved. SSF ethanol concentration were about 113 g/L at 1.5% PEG6000 addition. A technoeconomic feasibility showed a return on investment (ROI) of 8.13% using 0.5% PEG6000 for SHF (96 h) and 12.25% ROI for SSF control (72 h). Life cycle assessment for the various scenarios indicated higher environmental gains for best cases of SSF over SHF. The study shows the SSF approach (0% PEG6000; 72 h) facilitates higher process efficiencies; technoeconomic gains and high environmental sustainability for future scale-up and commercial realization.


Assuntos
Biocombustíveis , Etanol/metabolismo , Fermentação , Glucose/biossíntese , Triticum/metabolismo , Hidrólise , Polietilenoglicóis/metabolismo
8.
Bioresour Technol ; 282: 348-352, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30878886

RESUMO

In this present work nanocomposite composed of Mn-ZnO capped with Poly Ethylene Glycol (PEG) was utilized as heterogeneous catalyst for the transesterification of oil extracted from Nannochloropsis oculata into biodiesel using methanol as an acyl acceptor. The synthesized Mn-ZnO novel nanocomposite capped with Poly Ethylene Glycol (PEG) was characterized by using SEM and XRD. Lipid contents from the microalgae were extracted by sonication and biphasic solvent method. The process parameters involved for heterogeneous catalysis of N. oculata to biodiesel were optimized and found to be oil to methanol molar ratio of 1:15 (mol:mol), catalyst loading 3.5% (w/w) and reaction temperature of 60 °C for 4 h of reaction time by Response Surface Method. The reusability studies showed that the nano-catalyst can be reused efficiently for 4 cycles. The yield of biodiesel obtained from N. oculata species using Mn-ZnO nanocomposite capped with PEG was 87.5%.


Assuntos
Biocombustíveis , Metanol/metabolismo , Microalgas/metabolismo , Nanocompostos , Polietilenoglicóis/metabolismo , Estramenópilas/metabolismo , Catálise , Esterificação , Compostos de Manganês/química , Óxido de Zinco/química
9.
Nanoscale ; 11(11): 4970-4986, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30839018

RESUMO

Poor success rates and challenges associated with the current therapeutic strategies of inflammatory bowel disease (IBD) have accelerated the emergence of gene therapy as an alternative treatment option with great promise. However, oral delivery of nucleic acids (NAs) to an inflamed colon is challenged by multiple barriers presented by the gastrointestinal, extracellular and intracellular compartments. Therefore, we screened a series of polyaspartic acid-derived amphiphilic cationic polymers with varied hydrophobicity for their ability to deliver NAs into mammalian cells. Using the most effective TAC6 polymer, we then engineered biocompatible and stable nanogels composed of polyplexes (TAC6, NA) and an anionic polymer, sodium polyaspartate, that were able to deliver the NAs across mammalian cells using caveolae-mediated cellular uptake. We then utilized these nanogels for oral delivery of PIAS1 (protein inhibitor of activated STAT1), a SUMO 3 ligase, encoding plasmid DNA since PIAS1 is a key nodal therapeutic target for IBD due to its ability to control NF-κB-mediated inflammatory signaling. We show that plasmid delivery using TAC6-derived nanogels diminished gut inflammation in a murine colitis model. Therefore, our study presents engineering of orally deliverable nanogels that can target SUMOylation machinery to combat gut inflammation with very high efficacy.


Assuntos
Colite/terapia , Técnicas de Transferência de Genes/instrumentação , Terapia Genética/métodos , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Sumoilação , Administração Oral , Animais , Cátions/química , Linhagem Celular Tumoral , Colite/patologia , Colite/fisiopatologia , Colo/metabolismo , Colo/patologia , Colo/fisiopatologia , Modelos Animais de Doenças , Endocitose , Expressão Gênica , Terapia Genética/instrumentação , Humanos , Inflamação , Camundongos , Peptídeos/química , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polietilenoimina/química , Polietilenoimina/metabolismo , Polímeros/química , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo
10.
Carbohydr Polym ; 212: 142-149, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30832841

RESUMO

Mentha piperita essential oils (MPEO) were loaded into chitosan nanogel to use as antibiofilm agent against Streptococcus mutans and to protect its dental plaque. Chitosan nanoparticles (CsNPs) were prepared by sol-gel method using linking bridge of tripolyphosphate (TPP). Physiological properties of MPEO-CNs were assessed by FTIR, SEM/EDX, DLS and zeta potential. Release kinetics, MIC and MBC were determined for MPEO-CNs. Expression of biofilm-associated genes including 8 genes: grfB, C and D, brpA, spaP, gbpB, relA and vicR was investigated at the presence of sub-MIC of MPEO-CNs. Most abundant bioactive compounds of MPEO were l-menthol (45.05%) and l-menthal (17.53%). SEM/EDX exhibited successful entrapment of MPEO into CsNPs followed by the changes in abundance of elemental peaks. A signal at 1737 cm-1 on chitosan spectrum was attributed to the carboxylic (CO) groups overlapped by MPEO incorporation. A new signal at 2361 cm-1 was assigned to electrostatic interactions of amine groups in chitosan with phosphoric units of TPP within the MPEO-chitosan. MPEO incorporation into porous nanogel decreased monodispersity of the nanoparticles and then raises z-average. Maximum release of MPEO was about 50% during 360 h in a hydroalcoholic solvent at ambient temperature. The adherence of bacterial cells showed high sensitivity to the nanoformulation of MPEO compared with unloaded chitosan-nanogel. Antibiofilm inhibition of S. mutans occurred in 50 and 400 µg/mL for MPEO-CNs and unloaded-nanogel, respectively. Among biofilm synthesis genes, gtfB, gtfC, gtfD were slightly affected by MPEO-CNs treatment, while gbpB, spaP, brpA, relA, and vicR genes underwent significant down-regulation in the presence of both unloaded-nanogel and MPEO-loaded-nanogel. This study demonstrated that the MPEO-CNs promised an efficient nanoformulation with the greatest inhibitory action against some glycosyltransferase genes (gtfB, C and D) as important enzymes involved in extracellular polymers. Finally, the results concluded that MPEO-CNs have a potential use as antibiofilm agent in toothpaste or mouth washing formulations.


Assuntos
Biofilmes/efeitos dos fármacos , Quitosana/administração & dosagem , Óleos Vegetais/administração & dosagem , Polietilenoglicóis/administração & dosagem , Polietilenoimina/administração & dosagem , Streptococcus mutans/efeitos dos fármacos , Dente/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Quitosana/metabolismo , Cárie Dentária/tratamento farmacológico , Cárie Dentária/microbiologia , Placa Dentária/tratamento farmacológico , Placa Dentária/microbiologia , Humanos , Mentha piperita , Óleos Vegetais/isolamento & purificação , Óleos Vegetais/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoimina/metabolismo , Streptococcus mutans/crescimento & desenvolvimento , Dente/microbiologia
11.
PLoS Comput Biol ; 15(3): e1006775, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30840616

RESUMO

BACKGROUND: Thrombocytopenia is a major side-effect of cytotoxic cancer therapies. The aim of precision medicine is to develop therapy modifications accounting for the individual's risk. METHODOLOGY/PRINCIPLE FINDINGS: To solve this task, we develop an individualized bio-mechanistic model of the dynamics of bone marrow thrombopoiesis, circulating platelets and therapy effects thereon. Comprehensive biological knowledge regarding cell differentiation, amplification, apoptosis rates, transition times and corresponding regulations are translated into ordinary differential equations. A model of osteoblast/osteoclast interactions was incorporated to mechanistically describe bone marrow support of quiescent cell stages. Thrombopoietin (TPO) as a major regulator is explicitly modelled including pharmacokinetics and-dynamics of TPO injections. Effects of cytotoxic drugs are modelled by transient depletions of proliferating cells. To calibrate the model, we used population data from the literature and close-meshed individual data of N = 135 high-grade non-Hodgkin's lymphoma patients treated with CHOP-like chemotherapies. To limit the number of free parameters, several parsimony assumptions were derived from biological data and tested via Likelihood methods. Heterogeneity of patients was explained by a few model parameters. The over-fitting issue of individual parameter estimation was successfully dealt with a virtual participation of each patient in population-based experiments. The model qualitatively and quantitatively explains a number of biological observations such as the role of osteoblasts in explaining long-term toxic effects, megakaryocyte-mediated feedback on stem cells, bi-phasic stimulation of thrombopoiesis by TPO, dynamics of megakaryocyte ploidies and non-exponential platelet degradation. Almost all individual time series could be described with high precision. We demonstrated how the model can be used to provide predictions regarding individual therapy adaptations. CONCLUSIONS: We propose a mechanistic thrombopoiesis model of unprecedented comprehensiveness in both, biological mechanisms considered and experimental data sets explained. Our innovative method of parameter estimation allows robust determinations of individual parameter settings facilitating the development of individual treatment adaptations during chemotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Modelos Biológicos , Trombocitopenia/induzido quimicamente , Trombopoese/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Plaquetas/citologia , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Megacariócitos/citologia , Polietilenoglicóis/metabolismo , Células-Tronco/citologia , Trombopoetina/metabolismo , Fatores de Tempo
12.
Bioprocess Biosyst Eng ; 42(5): 807-815, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30707292

RESUMO

Co-polymerization of microbial polyesters, polyhydroxyalkanoates (PHAs), with synthetic polymers has become an established and promising tool in the recent past for improving the material and biological properties of the biopolyesters. Bacillus cereus RCL 02, a leaf endophytic bacterium of the oleaginous plant Ricinus communis L., has been reported to produce a significant amount of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] under batch cultivation. The present study demonstrates the synthesis and accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-polyethylene glycol [P(3HB-co-3HV)-PEG] co-polymer by the isolate RCL 02 in glucose, valeric acid, and PEG-200 supplemented mineral salts medium following dual-step cultivation. The identity of P(3HB-co-3HV)-PEG co-polymer so produced has been confirmed by X-ray diffraction (XRD) analysis, Fourier-transform infrared (FTIR), and proton nuclear magnetic resonance (1H NMR) spectroscopic studies, and the purified co-polymer was found to be composed of 3.2 mol% ethylene glycol (EG) and 8.4 mol% 3HV along with 3HB. While the thermogravimetric analysis (TGA) revealed that P(3HB-co-3HV)-PEG films degraded at 269.32 °C, differential scanning calorimetry (DSC) recorded the melting peak of the co-polymer at 163.8 °C. This study emphasized to explore the endophytic Bacillus spp. for production of P(3HB-co-3HV)-PEG co-polymers with improved material properties which may find possible application for biomedical purposes.


Assuntos
Bacillus cereus/metabolismo , Poliésteres/metabolismo , Polietilenoglicóis/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Poliésteres/química , Polietilenoglicóis/química , Poli-Hidroxialcanoatos/química
13.
Med Chem ; 15(6): 705-714, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30727907

RESUMO

BACKGROUND: L-asparaginase (L-ASN) is an anti-cancer enzyme therapeutic drug that exerts cytotoxicity via inhibition of protein synthesis through depletion of L-asparagine in the tumor microenvironment. The therapeutic performance of the native drug is partial due to the associated instability, reduced half-life and immunogenic complications. OBJECTIVE: In this study, we attempted the modification of recombinant L-asparaginase with PEG and an integrated computational strategy to probe the PEGylation in the protein to understand the biological stability/activity imparted by PEG. METHODS: In vitro PEGylation of recombinant L-ASN was carried out and further evaluated in silico. RESULTS: PEGylation enhanced thermal and pH activities with extended serum half-life and resistance to proteases compared to the native enzyme. The molecular dynamics analysis revealed intricate interactions required in the coupling of PEG to L-asparaginase to bestow stronger binding affinity of L-asparagine moiety towards L-asparaginase. PEG-asparagine complex ensured stable conformation over both the native protein and asparagine-protein complex thus elucidating the PEG-induced stable conformation in the protein. PEG mechanistically stabilized L-asparaginase through inducing pocket modification at the receptor to adapt to the cavity. CONCLUSION: The study provides the rationale of PEGylation in imparting the stability towards Lasparaginase which would expand the potential application of L-asparaginase enzyme for the effective treatment of cancer.


Assuntos
Antineoplásicos/química , Asparaginase/química , Polietilenoglicóis/química , Antineoplásicos/metabolismo , Asparaginase/metabolismo , Asparagina/metabolismo , Domínio Catalítico , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Polietilenoglicóis/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Estabilidade Proteica
14.
PLoS One ; 14(2): e0211951, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753228

RESUMO

L-asparaginase (ASNase) from Escherichia coli is currently used in some countries in its PEGylated form (ONCASPAR, pegaspargase) to treat acute lymphoblastic leukemia (ALL). PEGylation refers to the covalent attachment of poly(ethylene) glycol to the protein drug and it not only reduces the immune system activation but also decreases degradation by plasmatic proteases. However, pegaspargase is randomly PEGylated and, consequently, with a high degree of polydispersity in its final formulation. In this work we developed a site-specific N-terminus PEGylation protocol for ASNase. The monoPEG-ASNase was purified by anionic followed by size exclusion chromatography to a final purity of 99%. The highest yield of monoPEG-ASNase of 42% was obtained by the protein reaction with methoxy polyethylene glycol-carboxymethyl N-hydroxysuccinimidyl ester (10kDa) in 100 mM PBS at pH 7.5 and PEG:ASNase ratio of 25:1. The monoPEG-ASNase was found to maintain enzymatic stability for more days than ASNase, also was resistant to the plasma proteases like asparaginyl endopeptidase and cathepsin B. Additionally, monoPEG-ASNase was found to be potent against leukemic cell lines (MOLT-4 and REH) in vitro like polyPEG-ASNase. monoPEG-ASNase demonstrates its potential as a novel option for ALL treatment, being an inventive novelty that maintains the benefits of the current enzyme and solves challenges.


Assuntos
Asparaginase/química , Asparaginase/metabolismo , Polietilenoglicóis/metabolismo , Asparaginase/isolamento & purificação , Asparaginase/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Estabilidade Enzimática , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico
15.
Eur J Pharmacol ; 847: 103-112, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30710549

RESUMO

The effects on the vasculature produced by ethanol withdrawal include both vasodilatation and hypocontractility, although a detailed biochemical understanding of these processes is yet to be accomplished. Here, we sought to investigate some of the mechanisms underlying vascular hypocontractility induced by ethanol withdrawal. Male Wistar rats were treated with increasing doses of 3-9% ethanol (v/v) for 21 days and the impact of ethanol withdrawal on the vascular function was assessed 48 h after immediate ethanol suspension. Endothelium-denuded rat aortic rings showed a reduced contractile response to phenylephrine, angiotensin II, serotonin and KCl after ethanol withdrawal, but the same phenomenon was not observed in endothelium-intact rings. Indomethacin, but not L-NAME, tiron, PEG-catalase and SC560, restored the contractile response to phenylephrine of endothelium-denuded aortas from abstinent rats. Hyporeactivity to phenylephrine induced by ethanol withdrawal was reversed by SC236, a selective cyclooxygenase (COX)-2 inhibitor. Similarly, Ro1138452, a selective prostacyclin IP receptor antagonist, reversed vascular hypocontractility induced by ethanol withdrawal. Increased concentrations of 6-keto-prostaglandin (PG)F1α, a stable product of PGI2, was detected in endothelium-denuded aortas from abstinent rats, and this response was prevented by indomethacin. However, no changes in aortic PGE2 levels were detected after ethanol withdrawal. In situ quantification of hydrogen peroxide (H2O2) and nitric oxide (NO) using fluorescent dyes revealed that ethanol withdrawal decreased the levels of these two compounds in the tunica media. Our studies show that the vascular hypocontractility induced by ethanol withdrawal is independent of the endothelium and it is mediated by PGI2 derived from COX-2.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Epoprostenol/metabolismo , Etanol/efeitos adversos , Síndrome de Abstinência a Substâncias/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Compostos de Benzil/farmacologia , Catalase/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/farmacologia , Imidazóis/farmacologia , Indometacina/farmacologia , Masculino , Fenilefrina/farmacologia , Polietilenoglicóis/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Wistar , Sulfonamidas/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
16.
Cardiovasc Intervent Radiol ; 42(5): 751-762, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30761413

RESUMO

PURPOSE: To compare the accumulation and effect of liposomal doxorubicin in liver tissue treated by radiofrequency ablation (RFA) and irreversible electroporation (IRE) in in vivo porcine models. MATERIALS AND METHODS: Sixteen RFA and 16 IRE procedures were performed in healthy liver of two groups of three pigs. Multi-tined RFA parameters included: 100 W, target temperature 105°C for 7 min. 100 IRE pulses were delivered using two monopolar electrodes at 2250 V, 1 Hz, for 100 µsec. For each group, two pigs received 50 mg liposomal doxorubicin (0.5 mg/kg) as a drip infusion during ablation procedure, with one pig serving as control. Samples were harvested from the central and peripheral zones of the ablation at 24 and 72 h. Immunohistochemical analysis to evaluate the degree of cellular stress, DNA damage, and degree of apoptosis was performed. These and the ablation sizes were compared. Doxorubicin concentrations were also analyzed using fluorescence photometry of homogenized tissue. RESULTS: RFA treatment zones created with concomitant administration of doxorubicin at 24 h were significantly larger than controls (2.5 ± 0.3 cm vs. 2.2 ± 0.2 cm; p = 0.04). By contrast, IRE treatment zones were negatively influenced by chemotherapy (2.2 ± 0.4 cm vs. 2.6 ± 0.4 cm; p = 0.05). At 24 h, doxorubicin concentrations in peripheral and central zones of RFA were significantly increased in comparison with untreated parenchyma (0.431 ± 0.078 µg/g and 0.314 ± 0.055 µg/g vs. 0.18 ± 0.012 µg/g; p < 0.05). Doxorubicin concentrations in IRE zones were not significantly different from untreated liver (0.191 ± 0.049 µg/g and 0.210 ± 0.049 µg/g vs. 0.18 ± 0.012 µg/g). CONCLUSIONS: Whereas there is an increased accumulation of periprocedural doxorubicin and an associated increase in ablation zone following RFA, a contrary effect is noted with IRE. These discrepant findings suggest that different mechanisms and synergies will need to be considered in order to select optimal adjuvants for different classes of ablation devices.


Assuntos
Doxorrubicina/análogos & derivados , Eletroporação/métodos , Fígado/cirurgia , Ablação por Radiofrequência/métodos , Animais , Doxorrubicina/administração & dosagem , Doxorrubicina/metabolismo , Feminino , Modelos Animais , Polietilenoglicóis/metabolismo , Suínos
17.
Environ Toxicol ; 34(5): 561-572, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30786124

RESUMO

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray-form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1-10 µg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 µg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC-promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N-acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 µg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 µg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 µg/kg) and TEG (1000 µg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in BALF. These results suggest that TEG can potentiate BAC-induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray-form products containing this chemical combination is potentially harmful to humans.


Assuntos
Compostos de Benzalcônio/toxicidade , Lesão Pulmonar/induzido quimicamente , Pulmão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/induzido quimicamente , Polietilenoglicóis/toxicidade , Células A549 , Animais , Compostos de Benzalcônio/administração & dosagem , Compostos de Benzalcônio/metabolismo , Líquido da Lavagem Broncoalveolar/química , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Citocinas/análise , Sinergismo Farmacológico , Humanos , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Masculino , Estresse Oxidativo/imunologia , Pneumonia/metabolismo , Pneumonia/patologia , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/metabolismo , Ratos Sprague-Dawley
18.
Biotechnol Appl Biochem ; 66(3): 281-289, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30597637

RESUMO

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L-1  H-1 , respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg-1 , 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a KM value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.


Assuntos
Asparaginase/biossíntese , Asparaginase/química , Polietilenoglicóis/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Asparaginase/metabolismo , Humanos , Cinética , Polietilenoglicóis/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
19.
Chem Commun (Camb) ; 55(8): 1156-1159, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30632571

RESUMO

The diffusion of adequate peptide through an enzyme-embedded host hydrogel leads to the in situ start-up and growth of an interpenetrated fibrous network. Based on the enzyme-assisted self-assembly concept, both chemistry and mechanical features of the hybrid hydrogel can be tuned.


Assuntos
Fosfatase Alcalina/metabolismo , Difusão , Hidrogéis/metabolismo , Peptídeos/metabolismo , Hidrogéis/química , Estrutura Molecular , Tamanho da Partícula , Peptídeos/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Propriedades de Superfície
20.
Nanomedicine ; 17: 82-93, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659929

RESUMO

Macrophage recognition of nanoparticles is highly influenced by particle size and surface modification. Due to the lack of appropriate in vivo screening models, it is still challenging and time-consuming to characterize and optimize nanomedicines regarding this undesired clearance mechanism. Therefore, we validate zebrafish embryos as an emerging vertebrate screening tool to assess the macrophage sequestration of surface modified particulate formulations with varying particle size under realistic biological conditions. Liposomes with different PEG molecular weights (PEG350-PEG5000) at different PEG densities (3.0-10.0 mol%) and particle sizes between 60 and 120 nm were used as a well-established reference system showing various degrees of macrophage uptake. The results of in vitro experiments, zebrafish embryos, and in vivo rodent biodistribution studies were consistent, highlighting the validity of the newly introduced zebrafish macrophage clearance model. We hereby present a strategy for efficient, systematic and rapid nanomedicine optimization in order to facilitate the preclinical development of nanotherapeutics.


Assuntos
Lipossomos/metabolismo , Macrófagos/metabolismo , Polietilenoglicóis/metabolismo , Animais , Transporte Biológico , Feminino , Células Hep G2 , Humanos , Lipossomos/química , Lipossomos/farmacocinética , Modelos Animais , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ratos Wistar , Distribuição Tecidual , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo
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