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1.
Int J Nanomedicine ; 15: 3023-3038, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32431499

RESUMO

Introduction: Advanced tumor-targeted theranostic nanoparticles play a key role in tumor diagnosis and treatment research. In this study, we developed a multifunctional theranostic platform based on an amphiphilic hyaluronan/poly-(N-ε-carbobenzyloxy-L-lysine) derivative (HA-g-PZLL), superparamagnetic iron oxide (SPIO) and aggregation-induced emission (AIE) nanoparticles for tumor-targeted magnetic resonance (MR) and fluorescence (FL) dual-modal image-guided photodynamic therapy (PDT). Materials and Methods: The amphiphilic hyaluronan acid (HA) derivative HA-g-PZLL was synthesized by grafting hydrophobic poly-(N-ε-carbobenzyloxy-L-lysine) (PZLL) blocks onto hyaluronic acid by a click conjugation reaction. The obtained HA-g-PZLLs self-assembled into nanoparticles in the presence of AIE molecules and SPIO nanoparticles to produce tumor-targeted theranostic nanoparticles (SPIO/AIE@HA-g-PZLLs) with MR/FL dual-modal imaging ability. Cellular uptake of the theranostic nanoparticles was traced by confocal laser scanning microscopy (CLSM), flow cytometry and Prussian blue staining. The intracellular reactive oxygen species (ROS) generation characteristics of the theranostic nanoparticles were evaluated with CLSM and flow cytometry. The effect of PDT was evaluated by cytotoxicity assay. The dual-mode imaging ability of the nanoparticles was evaluated by a real-time near-infrared fluorescence imaging system and magnetic resonance imaging scanning. Results: The resulting theranostic nanoparticles not only emit red fluorescence for high-quality intracellular tracing but also effectively produce singlet oxygen for photodynamic tumor therapy. In vitro cytotoxicity experiments showed that these theranostic nanoparticles can be efficiently taken up and are mainly present in the cytoplasm of HepG2 cells. After internalization, these theranostic nanoparticles showed serious cytotoxicity to the growth of HepG2 cells after white light irradiation. Discussion: This work provides a simple method for the preparation of theranostic nanoparticles with AIE characteristics and MR contrast enhancement, and serves as a dual-modal imaging platform for image-guided tumor PDT.


Assuntos
Meios de Contraste/química , Imagem por Ressonância Magnética , Nanopartículas/química , Imagem Óptica , Fotoquimioterapia , Nanomedicina Teranóstica , Animais , Linhagem Celular Tumoral , Feminino , Fluorescência , Células Hep G2 , Humanos , Ácido Hialurônico/síntese química , Ácido Hialurônico/química , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/ultraestrutura , Polilisina/síntese química , Polilisina/química , Espectroscopia de Prótons por Ressonância Magnética , Espécies Reativas de Oxigênio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
2.
Int J Food Microbiol ; 328: 108663, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32454366

RESUMO

ε-Polylysine (ε-PL) is a natural and highly effective cationic antimicrobial, of which antibacterial activity is limited in food matrix because of ε-PL's charged amino groups that form complexes with food polyanions. Whey protein-ε-PL complexes delivery system was found to be able to solve the problem and keep the antibacterial activity. This study investigated the antibacterial activity of the complexes and its mechanism against Escherichia coli. The minimal inhibitory concentration of ε-PL was in the range 11.72-25.00 g/mL for the complexes containing different amount of ε-PL and was similar to that of free ε-PL. The results of scanning electron microscopy showed that the complexes could destroy the structure of E. coli cell membrane surface, leaving holes on the surface of the bacteria, leading to the death of the bacteria. The molecular dynamics simulation results showed that the mechanism of the antibacterial activity of the complexes was as follows: under electrostatic interaction, the complexes captured the phospholipid molecules of the bacterial membrane through the hydrogen bonds between the positively charged amino groups of ε-PL and the oxygen atom of the phosphate head groups of the membrane, which could create holes on the surface of the bacteria and lead to the death of the bacteria. The results of activity on real food systems showed that the complexes kept the number of E. coli within 5.8 log10 CFU/g after 7 d storage in sauced duck products, while the positive control (ε-PL) was 6.5 log10 CFU/g and negative control (sterile water) was 7.8 log10 CFU/g. Overall, this study confirmed the antibacterial activity of the complexes and provided fundamental knowledge of its antibacterial activity mechanism.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Polilisina/farmacologia , Proteínas do Soro do Leite/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Patos , Microbiologia de Alimentos , Ligação de Hidrogênio/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas do Soro do Leite/metabolismo
3.
PLoS One ; 15(5): e0232394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32365097

RESUMO

BACKGROUND: Astodrimer Gel contains a novel dendrimer intended to treat and prevent bacterial vaginosis. We assessed the efficacy and safety of Astodrimer Gel for treatment of bacterial vaginosis. METHODS: 132 women with bacterial vaginosis were randomized 1:1:1:1 to Astodrimer 0.5% (N = 34), 1% (N = 33), or 3% (N = 32) Gel or hydroxyethyl cellulose placebo gel (N = 33) at a dose of 5 g vaginally once daily for 7 days at 6 centers in the United States. The primary endpoint was clinical cure (no bacterial vaginosis vaginal discharge and no more than one of 1) vaginal pH ≥4.5; 2) ≥20% clue cells; or 3) positive whiff test) at study days 21-30. Secondary analyses included clinical cure at study days 9-12, patient-reported symptoms, acceptability and adverse events. RESULTS: The Astodrimer 1% Gel dose was superior to placebo for the primary and selected secondary efficacy measures in the modified intent-to-treat population. Clinical cure rates at day 9-12 were superior to placebo for the Astodrimer 3%, 1% and 0.5% Gel groups (62.5% [15/24; P = .002], 74.1% [20/27; P < .001], and 55.2% [16/29; P = .001], respectively, vs. 22.2% [6/27]). At day 21-30, clinical cure rates were 46.2% (12/26) for the 1% dose vs. 11.5% for placebo (3/26; P = .006). A greater proportion of patients reported absence of vaginal discharge and vaginal odor at day 9-12 and day 21-30 for Astodrimer Gel groups compared with placebo. Adverse events considered potentially treatment-related occurred in only 25% of Astodrimer Gel-treated patients vs. 22% of placebo patients. CONCLUSION: Astodrimer Gel once daily for 7 days was superior to placebo for treatment of bacterial vaginosis and was well-tolerated. The 1% dose consistently showed the strongest efficacy across endpoints. These results support a role for Astodrimer Gel, 1%, as an effective treatment for bacterial vaginosis.


Assuntos
Antibacterianos/administração & dosagem , Dendrímeros/administração & dosagem , Polilisina/administração & dosagem , Descarga Vaginal/tratamento farmacológico , Vaginose Bacteriana/tratamento farmacológico , Administração Intravaginal , Adulto , Antibacterianos/efeitos adversos , Dendrímeros/efeitos adversos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Géis , Humanos , Polilisina/efeitos adversos , Resultado do Tratamento , Adulto Jovem
4.
Cancer Immunol Immunother ; 69(8): 1651-1662, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32219501

RESUMO

BACKGROUND: This phase I study aimed to evaluate the safety, peptide-specific immune responses, and anti-tumor effects of a novel vaccination therapy comprising multi-HLA-binding heat shock protein (HSP) 70/glypican-3 (GPC3) peptides and a novel adjuvant combination of hLAG-3Ig and Poly-ICLC against metastatic gastrointestinal cancers. METHODS: HSP70/GPC3 peptides with high binding affinities for three HLA types (A*24:02, A*02:01, and A*02:06) were identified with our peptide prediction system. The peptides were intradermally administered with combined adjuvants on a weekly basis. This study was a phase I dose escalation clinical trial, which was carried out in a three patients' cohort; in total, 11 patients were enrolled for the recommended dose. RESULTS: Seventeen patients received this vaccination therapy without dose-limiting toxicity. All treatment-related adverse events were of grades 1 to 2. Peptide-specific CTL induction by HSP70 and GPC3 proteins was observed in 11 (64.7%) and 13 (76.5%) cases, respectively, regardless of the HLA type. Serum tumor marker levels were decreased in 10 cases (58.8%). Immunological analysis using PBMCs indicated that patients receiving dose level 3 presented with significantly reduced T cell immunoglobulin and mucin-domain containing-3 (TIM3)-expressing CD4 + T cells after one course of treatment. PD-1 or TIM3-expressing CD4 + T cells and T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT)-expressing CD8 + T cells in PBMCs before vaccination were negative predictive factors for survival. CONCLUSIONS: This novel peptide vaccination therapy was safe for patients with metastatic gastrointestinal cancers.


Assuntos
Carboximetilcelulose Sódica/análogos & derivados , Neoplasias Gastrointestinais/terapia , Glipicanas/imunologia , Antígenos HLA-A/imunologia , Antígenos HLA-G/administração & dosagem , Proteínas de Choque Térmico HSP70/imunologia , Fragmentos de Peptídeos/administração & dosagem , Poli I-C/administração & dosagem , Polilisina/análogos & derivados , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Carboximetilcelulose Sódica/administração & dosagem , Estudos de Coortes , Feminino , Seguimentos , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/metabolismo , Neoplasias Gastrointestinais/patologia , Glipicanas/metabolismo , Antígenos HLA-A/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Polilisina/administração & dosagem , Prognóstico , Taxa de Sobrevida
5.
PLoS One ; 15(2): e0222371, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023245

RESUMO

The study of cell aggregation in vitro has a tremendous importance these days. In cancer biology, aggregates and spheroids serve as model systems and are considered as pseudo-tumors that are more realistic than 2D cell cultures. Recently, in the context of brain tumors (gliomas), we developed a new poly(ethylene glycol) (PEG)-based hydrogel, with adhesive properties that can be controlled by the addition of poly(L-lysine) (PLL), and a stiffness close to the brain's. This substrate allows the motion of individual cells and the formation of cell aggregates (within one day), and we showed that on a non-adhesive substrate (PEG without PLL is inert for cells), the aggregates are bigger and less numerous than on an adhesive substrate (with PLL). In this article, we present new experimental results on the follow-up of the formation of aggregates on our hydrogels, from the early stages (individual cells) to the late stages (aggregate compaction), in order to compare, for two cell lines (F98 and U87), the aggregation process on the adhesive and non-adhesive substrates. We first show that a spaceless model of perikinetic aggregation can reproduce the experimental evolution of the number of aggregates, but not of the mean area of the aggregates. We thus develop a minimal off-lattice agent-based model, with a few simple rules reproducing the main processes that are at stack during aggregation. Our spatial model can reproduce very well the experimental temporal evolution of both the number of aggregates and their mean area, on adhesive and non-adhesive soft gels and for the two different cell lines. From the fit of the experimental data, we were able to infer the quantitative values of the speed of motion of each cell line, its rate of proliferation in aggregates and its ability to organize in 3D. We also found qualitative differences between the two cell lines regarding the ability of aggregates to compact. These parameters could be inferred for any cell line, and correlated with clinical properties such as aggressiveness and invasiveness.


Assuntos
Adesão Celular , Agregação Celular , Hidrogéis/química , Modelos Biológicos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Proliferação de Células , Humanos , Cinética , Polietilenoglicóis/química , Polilisina/química
6.
Int J Nanomedicine ; 15: 1205-1214, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32110017

RESUMO

Background: Celastrol (CEL), a triterpene extracted from the Chinese herb tripterygium wilfordii, has been reported to have profound anticancer activities. However, poor water solubility and high side toxicities have severely restricted the clinical applications of CEL. Purpose: We proposed a facile "in situ drug conjugation-induced self-assembly" strategy to prepare CEL-loaded nanoparticles (CEL-NPs) that exhibited enhanced antitumor activity against melanoma. Methods: First, the CEL was chemically conjugated onto a methoxyl poly(ethylene glycol)-b-poly(L-lysine) (mPEG-PLL) backbone, resulting in the conversion of the double hydrophilic mPEG-PLL polymer into an amphiphilic polymer prodrug, mPEG-PLL/CEL. The obtained mPEG-PLL/CEL could self-assemble into stable micelles in aqueous solution due to the hydrophobic association of CEL moieties in the side chains and the possible electrostatic interaction between the carboxyl group in CEL and the residue amine group in the PLL segment. Thus, the obtained mPEG-PLL/CEL nanoparticles were named CEL self-stabilized nanoparticles (CEL-NPs), which were then characterized by dynamic light scattering and transmission electron microscopy. Furthermore, the antitumor effects of the CEL-NPs were investigated by an MTT assay in vitro and in a B16F10 tumor-bearing mice model. Results: The CEL-NPs exhibited sustained drug release behavior and were effectively endocytosed by B16F10 cells. Furthermore, the in vivo antitumor evaluation demonstrated that the CEL-NPs had remarkably higher tumor growth inhibition rates and lower systemic side effects than free CEL. Conclusion: In summary, our present work not only demonstrates the generation of stable CEL-loaded nanoparticles for the efficient treatment of melanoma but also describes a general way to prepare drug self-stabilized nanomedicine for anticancer therapy.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Nanopartículas/química , Triterpenos/administração & dosagem , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacocinética , Preparações de Ação Retardada , Difusão Dinâmica da Luz , Interações Hidrofóbicas e Hidrofílicas , Masculino , Melanoma , Camundongos Endogâmicos C57BL , Micelas , Microscopia Eletrônica de Transmissão , Nanopartículas/administração & dosagem , Polietilenoglicóis/química , Polilisina/análogos & derivados , Polilisina/química , Triterpenos/farmacocinética
7.
J Biosci Bioeng ; 129(5): 558-564, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31924510

RESUMO

ε-Poly-l-lysine (ε-PL) produced as a secondary metabolite of Streptomyces albulus has long been used as a natural food preservative in a number of countries, including Japan, the United States, South Korea, and China. To date, numerous studies employing classical biotechnological approaches have been carried out to improve its productivity. Here we report a modern and rational genetic approach to enhancing metabolic flux toward ε-PL biosynthesis. Based on in silico genome analyses, we revealed that S. albulus NBRC14147 produces five antifungal polyene antibiotics-tetramycin A and B, tetrin A and B, and a trace amount of nystatin A1-concomitantly with antimicrobial ε-PL. Targeted inactivation of the biosynthetic gene cluster for tetramycins and tetrins in a nystatin A1 production-deficient mutant completely abolished the production of polyene macrolides, which in turn led to an approximately 20% improvement in ε-PL production that closely correlated with the polyene defects. The biosynthetic flux for ε-PL was thus successfully enhanced by inactivation of the concomitant secondary metabolite biosynthetic pathways. Since this elimination of concomitantly produced metabolites also allows for simpler purification after fermentation production of ε-PL, the rational strain engineering strategy we show here will improve its industrial production.


Assuntos
Macrolídeos/metabolismo , Polienos/metabolismo , Polilisina/biossíntese , Streptomyces/metabolismo , Fermentação , Conservantes de Alimentos/metabolismo , Macrolídeos/química , Polienos/química , Streptomyces/química , Streptomyces/genética
8.
Pestic Biochem Physiol ; 163: 147-153, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31973852

RESUMO

Microbial secondary metabolites produced by Streptomyces are applied to control plant diseases. ε-poly-l-lysine (ε-PL) is a non-toxic food preservative, but the potential application of ε-PL as a microbial fungicide in agriculture has rarely been reported. In this study, Alternaria alternata (A. alternata) was used to reveal the effect and mode of action for ε-PL on the plant pathogenic fungi. The results showed that ε-PL effectively inhibited necrotic-lesion development caused by A. alternata on tobacco. Mycelial growth was also significantly inhibited in vitro by 100 µg/ml ε-PL using in vitro analysis. Moreover, 25 µg/ml ε-PL inhibited spore germination and induced abnormal morphological development of A. alternata hyphae. To clarify the molecular-genetic antifungal mechanisms, we selected several crucial genes involved in the development and pathogenesis of A. alternata and studied their expression regulated by ε-PL. Results of real-time quantitative PCR showed that a mycelium morphology and pathogenic process related cyclic adenosine monophosphate protein (cAMP) dependent protein kinase A (PKA), Alternaria alternata cAMP-dependent protein kinase catalytic subunit (AAPK1) and the early infection-related glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were down-regulated after ε-PL treatment. The results provide novel insights for the application of ε-PL in the control of plant diseases caused by A. alternata.


Assuntos
Alternaria , Tabaco , Doenças das Plantas , Polilisina , Virulência
9.
J Agric Food Chem ; 68(4): 1101-1109, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31904947

RESUMO

ε-Poly-l-lysine (ε-PL) consists of 25-35 lysine residues which are linked by an isopeptide bond formed by dehydration condensation of α-carboxyl and ε-amino groups and has good antibacterial activity and broad-spectrum inhibition range. However, there is no clear conclusion about the structure and antibacterial mechanism of ε-PL in aqueous solution. Herein, a high purity of ε-PL was prepared using Amberlite IRC-50 ion-exchange resin. Membrane filtration and dynamic light scattering were used to study the variations of ε-PL aggregation in aqueous solution with pH value. The conformational changes and antibacterial activities of ε-PL and carbamoylated ε-PL in different water environments were studied with circular dichroism (CD) and inhibition zone. The structural changes during the spray-drying process were determined by Fourier transform infrared spectroscopy. The results indicated that the side chain amino charge played a decisive role in the ε-PL conformation and aggregation. ε-PL exhibited the properties of a ß-sheet during spray drying from acidic liquids to solids. The cation enhanced the antibacterial activity of ε-PL but did not play a key role. Instead, the backbone of ε-PL might determine the mechanism of ε-PL antibacterial.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Polilisina/química , Polilisina/farmacologia , Antibacterianos/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Transição de Fase , Polilisina/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Streptomyces/química , Streptomyces/metabolismo
10.
Food Chem Toxicol ; 136: 110935, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31693913

RESUMO

Silver nanoparticles (AgNPs) represent one of the most abundant biocidal nanomaterials contained in more than 30% of nano-enabled consumer products and 75% of nanomedical products. The cumulative exposure of the general population may therefore reach critical and potentially hazardous levels. Due to data gaps on AgNP effects in humans, it is urgent to further evaluate their possible toxicity, particularly in vulnerable systems like the nervous one. As AgNPs may cross the blood brain and placental barriers, this study evaluated the in vitro effect of different AgNPs on neuronal precursor cells. For this purpose, 10 nm-sized AgNPs were stabilized with five different coating agents rendering a neutral, positive and negative surface charge. Murine neural stem cells (mNSCs) were used as cellular model to test AgNP neurotoxicity by evaluating the range of toxicity endpoints including cellular viability, apoptosis induction, oxidative stress response, cellular and mitochondrial membrane damages, DNA damage, inflammation response, and neural stem cell regulation. Our results clearly showed that the neurotoxic potential of AgNPs was not dependent on their surface charge or coating agents used for their surface stabilization. All AgNP types exhibited significant toxicity in neuronal precursor cells at an in vitro dose of 5 mg Ag/L or lower.


Assuntos
Nanopartículas Metálicas/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Prata/toxicidade , Animais , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Cetrimônio/química , Cetrimônio/toxicidade , Dano ao DNA/efeitos dos fármacos , Ácido Dioctil Sulfossuccínico/química , Ácido Dioctil Sulfossuccínico/toxicidade , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Polilisina/química , Polilisina/toxicidade , Povidona/química , Povidona/toxicidade , Soroalbumina Bovina/química , Soroalbumina Bovina/toxicidade , Prata/química , Transcriptoma/efeitos dos fármacos
11.
Biochim Biophys Acta Biomembr ; 1862(2): 183128, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734310

RESUMO

Clusters of positively-charged basic amino acid residues, particularly lysine, are known to promote the interaction of many peripheral membrane proteins with the cytoplasmic surface of the plasma membrane via electrostatic interactions. In this work, cholesterol's effects on the interaction between lysine residues and membranes have been studied. Using poly-l-lysine (PLL) and vesicles as models to mimic the interaction between lysine-rich protein domains and the plasma membrane, light scattering measurements indicated cholesterol enhanced the electrostatic interaction through indirectly affecting the negatively charged phospholipid dioleoylphosphatidylserine, DOPS. Addition of PLL to lipid vesicles containing DOPS showed an initial increase in static light scattering (SLS), attributed to binding of PLL to the vesicle surface, followed by a slower continuously declining SLS signal, which, from comparison with fluorescent dye leakage studies could be attributed to vesicle lysis. Although electrostatic interactions between PLL and the membrane were not necessary for penetration to occur, cholesterol promoted membrane disruption of negatively charged vesicles, possibly by increasing the electrostatic interactions between PLL and the membrane. In contrast, cholesterol lowered the susceptibility of uncharged vesicles (formed using dioleoylphosphatidylcholine, DOPC) to PLL penetration. This can be explained by the absence of electrostatic interactions and cholesterol's known ability to increase membrane thickness and mechanical strength. Thus, the ability of cationic peptides to penetrate membranes including cholesterol is likely to depend on the membrane's PS:PC ratio.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Fosfatidilserinas/química , Polilisina/química , Permeabilidade da Membrana Celular , Bicamadas Lipídicas/metabolismo , Polilisina/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-31676442

RESUMO

Pancreatic lipase (PNLIP) is a digestive enzyme that is a potential drug target for the treatment of obesity. A better understanding of its regulation mechanisms would facilitate the development of new therapeutics. Recent studies indicate that intestinal lipolysis by PNLIP is reduced by Angiopoietin-like protein 4 (ANGPTL4), whose N-terminal domain (nANGPTL4) is a known inactivator of lipoprotein lipase (LPL) in blood circulation and adipocytes. To elucidate the mechanism of PNLIP inhibition by ANGPTL4, we developed a novel approach, using isothermal titration calorimetry (ITC). The obtained results were compared with those of well-described inhibitors of PNLIP - ε-polylysine (EPL), (-)-epigallocatechin-3-gallate (EGCG) and tetrahydrolipstatin. We demonstrate that ITC allows to investigate PNLIP inhibition mechanisms in complex substrate emulsions and that the ITC-based assay is highly sensitive - the lowest concentration for quantification of PNLIP is 1.5 pM. Combining ITC with surface plasmon resonance and fluorescence measurements, we present evidence that ANGPTL4 is a lipid-binding protein that influences PNLIP activity through interactions with components of substrate emulsions (bile salts, phospholipids and triglycerides), and this promotes the aggregation of triglyceride emulsions similarly to the PNLIP inhibitors EPL and EGCG. In the absence of substrate emulsion, unlike in the case of LPL, ANGPTL4 did not induce the inactivation of PNLIP. Our data also prove that due to various interactions with components of substrate systems, the effect of a PNLIP inhibitor depends on whether its effect is measured in a complex substrate emulsion or in a simple substrate system.


Assuntos
Proteína 4 Semelhante a Angiopoietina/farmacologia , Fármacos Antiobesidade/farmacologia , Calorimetria , Ensaios Enzimáticos/métodos , Lipase/antagonistas & inibidores , Proteína 4 Semelhante a Angiopoietina/uso terapêutico , Fármacos Antiobesidade/uso terapêutico , Catequina/análogos & derivados , Catequina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Lipase/genética , Lipase/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Orlistate/farmacologia , Polilisina/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Talanta ; 208: 120445, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31816708

RESUMO

Multi-channel capillaries (MC) formed from thousands individual microcapillaries with diameters ranging 10-100 µm are of a great interest for their use as platforms for molecular imprinting due to their relatively large surface area, high mechanical stability and possibility of facile integration in sensor systems. The manuscript proposes a new format of immunoassay based on imprinted protein immobilized on a MC inner surface modified with poly-l-lysine. The combination of the environmentally friendly, easy-to-produce and cheap recognition element with the carrier allowing to increase the assay sensitivity makes the described technique a perspective alternative for the existing screening tests. Two bioimprinting approaches were described. The imprinted protein (ovalbumin, OVA) primarily prepared separately and later immobilized on a MC structure was compared to the imprinted OVA directly prepared on the MC surface. Detection of a food contaminant zearalenone was chosen as a proof-of-concept. In a case of the immobilization of the primarily prepared imprinted OVA the reached limit of detection (LOD) was 0.8 ng/mL, and for the in-situ imprinted OVA the LOD was 0.12 ng/mL. The sensitivity of the developed bioimprinted assay was comparable to the commercially available ELISA kits for ZEN detection. The OVA in-situ imprinted on the MC surface was tested for the detection of ZEN in artificially spiked wheat samples. The high recovery values (88-112%) and good repeatability (RSD of 8.5-9.6%) were demonstrated allowing to conclude that the IPs-based MC-ELISA is a promising tool for analysis of the mycotoxin in complex matrices.


Assuntos
Contaminação de Alimentos/análise , Imunoensaio/métodos , Impressão Molecular , Ovalbumina/química , Triticum/química , Zearalenona/análise , Vidro , Peroxidase do Rábano Silvestre/química , Proteínas Imobilizadas/química , Polilisina/química , Soroalbumina Bovina/química , Zearalenona/química
14.
Mater Sci Eng C Mater Biol Appl ; 106: 110251, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753346

RESUMO

In this work, a versatile folic acid (FA) decorated reductive-responsive ε-poly-l-Lysine (ε-PL)-based microcapsules (FA-ε-PLMCs) were designed and facilely assembled by using sonochemical technique. Cellular uptake experiment of FA-ε-PLMCs loaded with Coumarin 6 (C6) as a model of hydrophobic drugs implied that hydrophobic drugs encapsulated inside FA-ε-PLMCs could be delivered selectively into Hela cells via folate-receptor (FR)-mediated endocytosis due to FA decorated on microcapsules. Furthermore, the shells of FA-ε-PLMCs cross-linked by disulfide bonds were derived from sulfhydryl groups (-SH) under ultrasonication. Under reductive environment, the hydrophobic drugs loaded in FA-ε-PLMCs would be easily released due to the cleavage of disulfide bonds. Benefiting from their suitable particle size, good loading capacity for hydrophobic drugs, remarkable targetability and reductive-triggered release, the obtained FA-ε-PLMCs could be a promising hydrophobic drugs carrier for the cancer treatment.


Assuntos
Cápsulas/química , Portadores de Fármacos/química , Ácido Fólico/química , Polilisina/química , Sistemas de Liberação de Medicamentos/métodos , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Espectroscopia de Infravermelho com Transformada de Fourier
15.
Mater Sci Eng C Mater Biol Appl ; 107: 110212, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761208

RESUMO

A critical challenge to the development of tissue engineering small-diameter vascular grafts is to achieve rapid endothelialization and long-term anticoagulation. It is necessary to graft both adhesion and antithrombus factors onto the surface of polycaprolactone without burst release to promote endothelial cell affinity and antithrombogenicity. A bionic structure with a nanocoating that allows a biologically responsive, long-term release was employed in this work to enable the grafting of various bioactive molecules such as gelatin, polylysine, and heparin. This approach involved orienting the biomimetic vascular structures; the self-assembly grafting of gelatin, polylysine, and heparin nanoparticles; and genipin crosslinking to form a multiphase crosslinked nanocoating. In this biologically inspired design, vascular endothelialization and long-term anticoagulation were successfully induced through a matrix metallopeptidase 2 regulative mechanism by delivering both adhesion and antithrombus factors with a responsive, long-term release without burst release. The method provided a simple and effective approach for delivering dual factors for tissue engineering small-diameter vascular grafts.


Assuntos
Materiais Biomiméticos/química , Nanotecnologia , Materiais Biomiméticos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/citologia , Prótese Vascular , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Gelatina/química , Heparina/química , Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Nanofibras/química , Poliésteres/química , Polilisina/química , Propriedades de Superfície
16.
Carbohydr Polym ; 229: 115484, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826482

RESUMO

A novel chitosan-based multifunctional nanoparticle (PY-CS-PLA) using cationic polylysine (PL) polymer and L-cysteine has been developed and investigated for the oral delivery of paclitaxel (PTX). As amphiphilic polymer, PY-CS-PLA presented good capability in self-assembling into spherical nanoparticle with mean size of 165 nm, and encapsulating PTX into the hydrophobic core. The encapsulated PTX was observed to be sustainedly released from the functionalized chitosan nanoparticle, and with a positive correlation to the pH value of the medium in the range of 1.2 to 7.4. The in vitro studies indicated that PY-CS-PLA/PTX could effectively enhance the cellular uptake of the PTX in Caco-2 cells. Pharmacokinetic result indicated that the oral bioavailability of PY-CS-PLA/PTX in rats was determined to be 5.63-fold to that of Taxol. Moreover, PY-CS-PLA/PTX improved the distribution of PTX in tumor site and presented better antitumor efficacy in Heps tumor-bearing mice and with less toxicity than other formulations. In conclusion, the PY-CS-PLA/PTX nanoparticle might be developed as a promising delivery vehicle for improving the oral bioavailability and therapeutic effect of hydrophobic antitumor drugs.


Assuntos
Quitosana/química , Cisteína/química , Portadores de Fármacos/química , Nanopartículas/química , Paclitaxel/administração & dosagem , Paclitaxel/química , Polilisina/química , Administração Oral , Animais , Células CACO-2 , Humanos , Camundongos , Paclitaxel/farmacocinética , Paclitaxel/farmacologia , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Nat Commun ; 10(1): 5439, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784506

RESUMO

Programmable behavior combined with tailored stiffness and tunable biomechanical response are key requirements for developing successful materials. However, these properties are still an elusive goal for protein-based biomaterials. Here, we use protein-polymer interactions to manipulate the stiffness of protein-based hydrogels made from bovine serum albumin (BSA) by using polyelectrolytes such as polyethyleneimine (PEI) and poly-L-lysine (PLL) at various concentrations. This approach confers protein-hydrogels with tunable wide-range stiffness, from ~10-64 kPa, without affecting the protein mechanics and nanostructure. We use the 6-fold increase in stiffness induced by PEI to program BSA hydrogels in various shapes. By utilizing the characteristic protein unfolding we can induce reversible shape-memory behavior of these composite materials using chemical denaturing solutions. The approach demonstrated here, based on protein engineering and polymer reinforcing, may enable the development and investigation of smart biomaterials and extend protein hydrogel capabilities beyond their conventional applications.


Assuntos
Materiais Biocompatíveis , Hidrogéis/metabolismo , Nanoestruturas , Polietilenoimina/metabolismo , Polilisina/metabolismo , Desdobramento de Proteína , Soroalbumina Bovina/metabolismo , Animais , Fenômenos Biomecânicos , Bovinos , Elasticidade , Hidrogéis/química , Soroalbumina Bovina/química
18.
Soft Matter ; 15(48): 9931-9941, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31764921

RESUMO

Geometrical features play a very important role in neuronal growth and the formation of functional connections between neuronal cells. Here, we analyze the dynamics of axonal growth for neuronal cells cultured on micro-patterned polydimethylsiloxane surfaces. We utilize fluorescence microscopy to image axons, quantify their dynamics, and demonstrate that periodic geometrical patterns impart strong directional bias to neuronal growth. We quantify axonal alignment and present a general stochastic approach that quantitatively describes the dynamics of the growth cones. Neuronal growth is described by a general phenomenological model, based on a simple automatic controller with a closed-loop feedback system. We demonstrate that axonal alignment on these substrates is determined by the surface geometry, and it is quantified by the deterministic part of the stochastic (Langevin and Fokker-Planck) equations. We also show that the axonal alignment with the surface patterns is greatly suppressed by the neuron treatment with Blebbistatin, a chemical compound that inhibits the activity of myosin II. These results give new insight into the role played by the molecular motors and external geometrical cues in guiding axonal growth, and could lead to novel approaches for bioengineering neuronal regeneration platforms.


Assuntos
Dimetilpolisiloxanos , Neurogênese , Neurônios/fisiologia , Polilisina , Animais , Células Cultivadas , Microscopia de Força Atômica , Microscopia de Fluorescência , Ratos
19.
Mikrochim Acta ; 186(12): 842, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31768653

RESUMO

Conjugated polymer hybrid nanoparticles (NPs) loaded with both indocyanine green (ICG) and 1,3-diphenylisobenzofuran (DPBF) are described. The NPs are dually functional in that ICG acts as the photosensitizer, and DPBF as a probe for singlet oxygen (1O2 probe). The nanoparticle core consists of the energy donating host poly(9,9-dioctylfluorenyl-2,7-diyl)-co-(2,5-p-xylene) (PFP). The polymer is doped with the energy acceptor DPBF. Ratiometric fluorometric detection of singlet oxygen is accomplished by measurement of fluorescence at wavelengths of 415 and 458 nm. In addition, the shell of the positively charged polymeric nanoparticles was modified, via electrostatic interaction, with negatively charged PDT drugs ICG. The integrated nanoparticles of type ICG-DPBF-PFP display effective photodynamic performance under 808-nm laser irradiation. The 1O2 sensing behaviors of samples are evaluated based on the ratiometric fluorescent responses produced by DPBF and PFP. 1O2 can be fluorimetically sensed with a detection limit of 28 µM. The multifunctional nanoprobes exhibit effortless cellular uptake, superior photodynamic activity and a rapid ratiometric response to 1O2. Graphical abstractSchematic of a dual-functional nanoplatform for photodynamic therapy (PDT) and singlet oxygen (1O2) feedback. It offers a new strategy for self-monitoring photodynamic ablation. FRET: fluorescence resonance energy transfer. Indocyanine green is attached in the shell of nanoparticles, and 1,3-diphenylisobenzofuran is doped into the energy donating host conjugated polymer.


Assuntos
Benzofuranos/química , Verde de Indocianina/química , Nanopartículas/química , Fármacos Fotossensibilizantes/química , Polilisina/química , Oxigênio Singlete/análise , Benzofuranos/toxicidade , Transferência Ressonante de Energia de Fluorescência , Células Hep G2 , Humanos , Verde de Indocianina/efeitos da radiação , Verde de Indocianina/toxicidade , Raios Infravermelhos , Limite de Detecção , Nanopartículas/toxicidade , Fotoquimioterapia , Fármacos Fotossensibilizantes/efeitos da radiação , Fármacos Fotossensibilizantes/toxicidade , Polilisina/toxicidade , Oxigênio Singlete/química
20.
Biomed Res Int ; 2019: 5478369, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781622

RESUMO

Vascular stent interventional therapy is the main method for clinical treatment of coronary artery diseases. However, due to the insufficient biocompatibility of cardiovascular materials, the implantation of stents often leads to serious adverse cardiac events. Surface biofunctional modification to improve the biocompatibility of vascular stents has been the focus of current research. In this study, based on the structure and function of extracellular matrix on vascular injury healing, a novel fibronectin-loaded poly-l-lysine/heparin nanoparticles was constructed for stent surface modification. In vitro blood compatibility evaluation results showed that the nanoparticles-modified surface could effectively reduce platelet adhesion and activation. In vitro cellular compatibility evaluation results indicated that the nanocoating may provide adequate efficacy in promoting the adhesion and proliferation of endothelial cells and thereby accelerate endothelialization. This study provides a new approach for the surface biological function modification of vascular stents.


Assuntos
Plaquetas/metabolismo , Materiais Revestidos Biocompatíveis/química , Células Endoteliais/metabolismo , Teste de Materiais , Neointima/metabolismo , Adesividade Plaquetária , Polieletrólitos/química , Stents , Adesão Celular , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/cirurgia , Fibronectinas , Heparina/química , Humanos , Neointima/patologia , Polilisina/química
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