RESUMO
The interaction of lipopolysaccharide with a recombinant protein is a serious bottleneck, particularly in the purification step of bioprocessing. Recombinant hepatitis B surface antigen (rHBsAg), the active ingredient of the hepatitis B vaccine, is probably contaminated by extrinsic LPS like other biopharmaceuticals. This research intends to eliminate LPS from its mixture with rHBsAg efficiently. Immobilized polymyxin B on magnetic nanoparticles (PMB-MNPs) was synthesized and implemented as an enhanced LPS affinity adsorbent (LAA). The 20-80 EU/dose binary samples with and without surfactant were applied to PMB-MNPs. Formerly, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were examined on the samples to qualitatively show the dissociation effect of the surfactant. Considering the high potential interaction of LPS with HBsAg, the dissociation effects of 0.5 and 1.5% Tween 20 on the binary samples were assessed using immunoaffinity chromatography (IAC) as a quantification tool. The dissociation effect of Tween 20 substantially diminished the interaction, leading to a proportional increase of free LPS up to 66%. The synergetic effect of Tween 20 and privileged LAA was highly effective in eliminating more than 80% of LPS with a remarkable LPS clearance factor of 5.8 and a substantial protein recovery rate of 97%.
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Despite the widespread use of silver nanoparticles (NPs), these NPs can accumulate and have toxic effects on various organs. However, the effects of silver nanostructures (Ag-NS) with alginate coating on the male reproductive system have not been studied. Therefore, this study aimed to investigate the impacts of this NS on sperm function and testicular structure. After the synthesis and characterization of Ag-NS, the animals were divided into five groups (n = 8), including one control group, two sham groups (received 1.5 mg/kg/day alginate solution for 14 and 35 days), and two treatment groups (received Ag-NS at the same dose and time). Following injections, sperm parameters, apoptosis, and autophagy were analyzed by the TUNEL assay and measurement of the mRNA expression of Bax, Bcl-2, caspase-3, LC3, and Beclin-1. Fertilization rate was assessed by in vitro fertilization (IVF), and testicular structure was analyzed using the TUNEL assay and hematoxylin and eosin (H&E) staining. The results showed that the NS was rod-shaped, had a size of about 60 nm, and could reduce sperm function and fertility. Gene expression results demonstrated an increase in the apoptotic markers and a decrease in autophagy markers, indicating apoptotic cell death. Moreover, Ag-NS invaded testicular tissues, especially in the chronic phase (35 days), resulting in tissue alteration and epithelium disintegration. The results suggest that sperm parameters and fertility were affected. In addition, NS has negative influences on testicular tissues, causing infertility in men exposed to these NS.
RESUMO
Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.
Assuntos
Cromatografia de Afinidade , Antígenos de Superfície da Hepatite B , Lipopolissacarídeos , Proteínas Recombinantes , Lipopolissacarídeos/análise , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/ultraestrutura , Microscopia Eletrônica de Transmissão , Vacinas contra Hepatite B/química , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/isolamento & purificação , Modelos Químicos , Sequência de Aminoácidos , Difusão Dinâmica da Luz , Cromatografia de Afinidade/métodosRESUMO
Today, bacterial cellulose has received a great deal of attention for its medical applications due to its unique structural properties such as high porosity, good fluid uptake, good strength, and biocompatibility. This study aimed to fabricate and study bacterial cellulose/graphitic carbon nitride/nettles/trachyspermum nanocomposite by immersion and PVA/BC/g-C3 N4 /nettles/trachyspermum nanofiber by electrospinning method as a wound dressing. The g-C3 N4 and g-C3 N4 solution were synthesized and then were characterized using Fourier transform infrared, X-ray diffraction, Zeta Potential, and scanning electronic microscope analyzes. Also, the antibacterial properties of the synthesized materials were proved by gram-positive and gram-negative bacteria using the minimum inhibitory concentration method. Besides, the toxicity, migration, and cell proliferation results of the synthesized materials on NIH 3T3 fibroblasts were evaluated using MTT and scratch assays and showed that the BC/PVA/g-C3 N4 /nettles/trachyspermum composite not only had no toxic effect on cells but also contributed to cell survival, cell migration, and proliferation has done. To evaluate the mechanical properties, a tensile strength test was performed on PVA/BC/g-C3 N4 /nettles/trachyspermum nanofibers, and the results showed good strength of the nanocomposite. In addition, in vivo assay, the produced nanofibers were used to evaluate wound healing, and the results showed that these nanofibers were able to accelerate the wound healing process so that after 14 days, the wound healing percentage showed 95%. Therefore, this study shows that PVA/BC/g-C3 N4 /nettles/trachyspermum nanofibers effectively inhibit bacterial growth and accelerate wound healing.
Assuntos
Antibacterianos , Bandagens , Celulose , Grafite , Compostos de Nitrogênio , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Apiaceae/química , Bactérias/química , Bactérias/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Celulose/química , Celulose/farmacologia , Grafite/química , Grafite/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Células NIH 3T3 , Nanofibras/química , Compostos de Nitrogênio/química , Compostos de Nitrogênio/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Álcool de Polivinil/química , Stachys/químicaRESUMO
A type of multi-sensitive ABC-CBA block copolymer with thermal, glutathione and pH-responsive bonds was synthesized via ring opening polymerization along with cationic ring opening mechanisms. In continuum, the synthesized copolymer strands self-assembled into nanomicelles. The linear copolymer is comprised poly (methoxy ethylene glycol)-b-poly (2-ethyl-2-oxazoline)-b-poly (ε-caprolactone)-cystamine (i.e. [mPEG-b-PEtOz-PCL]2-Cys) and the curcumin was encapsulated inside the micelles mostly through hydrophobic interaction. The H-NMR, FTIR and GPC analysis were applied to identify the composition structure of the copolymer. The critical micelle concentration (CMC) value was achieved favorably 0.01 mg/mL for the synthesized copolymer. The morphology and particle size of solid nanocarrier were characterized by DLS, Zeta potential, AFM, TEM, and SEM micrographs. The drug loading content for the curcumin was attained 13.3% (w/w), and the entrapment efficacy of the drug in nanocarrier was obtained 79 percent. The in vitro release profile of the drug-loaded micelle was investigated by exposure to different pH, temperature and reduction circumstances, stimulated by tumor microenvironment conditions. The cell viability assay of the drug-loaded nanocarrier demonstrates high cytotoxicity toward HDF cells, while the drug-free nanocarrier has trifling toxicity and good biocompatibility. Therefore, according to the pleasant output of the research, this novel nanomicelle based on ABC-CBA block copolymer can be carried out effectively as an efficient nanocarrier in targeted drug delivery.
Assuntos
Curcumina , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Micelas , Tamanho da Partícula , Poliésteres , Polietilenoglicóis , PolímerosRESUMO
BACKGROUND: Some male survivors of childhood cancer are suffering from azoospermia. In addition, spermatogonial stem cells (SSCs) are necessary for the improvement of spermatogenesis subsequent to exposure to cytotoxic agents such as cisplatin. OBJECTIVE: The aim of this study was to evaluate the anticancer activity of cisplatin-loaded folic acid-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on mouse malignant cell line (EL4) and SSCs in vitro. METHODS: SSCs were co-cultured with mouse malignant cell line (EL4) cells and divided into four culture groups: 1) control (cells were co-cultured in the culture medium), 2) co-cultured cells were treated with cisplatin (10 µg/mL), 3) co-cultured cells were treated with cisplatin-loaded folic acid-conjugated PLGA NPs, and 4) co-cultures were treated with folic acid-conjugated PLGA for 48 hours. The NPs were prepared, characterized, and targeted with folate. In vitro release characteristics, loading efficiency, and scanning electron microscopy and transmission electron microscopy images were studied. Cancer cells were assayed after treatment using flow cytometry and TUNEL assay. The co-cultures of SSCs and EL4 cells were injected into seminiferous tubules of the testes after treating with cis-diaminedichloroplatinum/PLGA NPs. RESULTS: The mean diameter of PLGA NPs ranged between 150 and 250 nm. The number of TUNEL-positive cells increased, and the expression of Bax and caspase-3 were upregulated in EL4 cells in Group 4 compared with Group 2. There was no pathological tumor in testes after transplantation with treated co-cultured cells. CONCLUSION: The PLGA NPs appeared to act as a promising carrier for cisplatin administration, which was consistent with a higher activation of apoptosis than free drug.
Assuntos
Antineoplásicos/administração & dosagem , Cisplatino/administração & dosagem , Ácido Fólico/química , Nanopartículas/administração & dosagem , Espermatogônias/patologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Técnicas de Cocultura , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Ácido Fólico/administração & dosagem , Ácido Láctico/química , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/patologia , Espermatogônias/efeitos dos fármacosRESUMO
Biomedical application of human pluripotent stem cell-derived hepatocyte-like cells (hPSC-HLCs) relies on efficient large-scale differentiation, which is commonly performed by a suspension culture of three-dimensional (3D) multicellular spheroids in bioreactors. However, this approach requires large amounts of growth factors (GFs) and the need to overcome limited diffusional transport posed by the inherent 3D structure of hPSC spheroids. Here, we have hypothesized that localized delivery of GFs by incorporation of GF-laden degradable polymeric microparticles (MPs) within the hPSC spheroids would circumvent such limitations. In this study, GFs for hepatocytic differentiation were encapsulated in gelatin-coated poly (l-lactic acid)/poly (DL-lactic-co-glycolic acid) (PLLA/PLGA) MPs which were subsequently incorporated into the hPSC spheroids. Gene expression analyses demonstrated that MP delivery of the GFs resulted in similar expression levels of hepatocytic markers despite the use of 10-fold less total GFs. The differentiated HLCs in the MP group exhibited ultrastructure and functional characteristics comparable with the conventional soluble GF group. The generated HLCs in the MP group were successfully engrafted in an acute liver injury mouse model and maintained hepatocytic function after implantation. These results suggested that sustained and localized delivery of GFs using MPs might offer a novel approach towards scalable technologies for hepatocytic differentiation and engineer a better 3D microenvironment for cells.
Assuntos
Técnicas de Cultura de Células/métodos , Micropartículas Derivadas de Células/química , Hepatócitos/citologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Reatores Biológicos , Diferenciação Celular/fisiologia , Imunofluorescência , Humanos , Masculino , Camundongos , Esferoides Celulares/citologiaRESUMO
Self-assembled nanomicelles can be used as synthetic biomaterials and colloidal carriers for poorly water-soluble drug delivery systems. Some of these micellar systems have been introduced in clinical trials and showed hopeful results relating to their therapeutic index in patients. Biodegradable nanomicelle was prepared from self-assembling amphiphilic block copolymer composed of poly(DL-lactic-co-glycolic acid) (PLGA) as a core and polyethylene glycol (PEG) as a corona. The PLGA-PEG block copolymer was first synthesized and characterized by FTIR, (1)H NMR, GPC and inherent viscosity measurements. The nanomicelle formed by PLGA-PEG block copolymer in the aqueous solution was characterized by dynamic light scattering, zeta potential, scanning electron microscopy (SEM) and fluorescence excitation and emission spectra of pyrene probe. The critical micelle concentration of obtained nanomicelle was about 0.006 mg/mL, with the size of about 160 nm and the zeta potential of -29 mV. Insulin-loaded PLGA-PEG nanomicelles were prepared by modified dialysis method and the physicochemical parameters of the micelles such as drug content, entrapment efficiency and in vitro drug release were characterized. The results showed that insulin was entrapped into PLGA-PEG nanomicelles with drug loading of 3.9 wt% and entrapment efficiency of 55 wt%. The nanomicelles containing insulin exhibited a controlled release profile. These observations suggested that the PLGA-PEG block copolymers nanomicelles have been prepared by a new synthetic route are potent nanocarrier for poorly water-soluble drugs as insulin.
Assuntos
Insulina/administração & dosagem , Ácido Láctico/química , Polietilenoglicóis/química , Ácido Poliglicólico/química , Portadores de Fármacos , Espectroscopia de Ressonância Magnética , Micelas , Microscopia Eletrônica de Varredura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Copoly(styrene-butyl acrylate-ethyleneglycoldimethacrylate) (St-BA-EGDMA) nanoparticles were prepared using miniemulsion polymerization technique. Then the dispersed nanoparticles in DMAc were added to in situ condensation polymerization media of pyromellitic dianhydride (PMDA) and oxydianiline (ODA) and consequently, homogenous polyamic acid solution containing the nanoparticles was obtained. Novel polymer-polymer nanocomposites were prepared by casting of PMDA-ODA polyamic acid solution with various content of the above elastomeric nanoparticle (ENP) on a glass plate followed by thermal imidization. All samples were characterized after preparation by FT-IR spectroscopy, transition electron microscopy (TEM), thermal gravimetry analysis (TGA) and differential scanning calorimetery (DSC). To investigate the adhesion strength of polyimides filled with (St-BA-EGDMA) nanoparticles, lap shear strength (LSS) test was examined on different metallic surfaces. Effect of nanoparticles content on the adhesion properties of this polymer was considerable for aluminum surface. Lap-shear strength and adhesive energy of the bonded samples were found to initially increase with the increase in ENP wt%, but decrease after a critical value. It was shown that by increasing the nanoparticles amount up to 25 wt%, the adhesion strength of polyimides increased due to the good wetability of surfaces. After that and by increasing the nanoparticles amount, the adhesion strength decreased according to the diminished strength between polyimide chains. Scanning Electron Microscopy (SEM) micrographs of the fractured surfaces were taken to determine the failure mode. They showed that by increasing the nanoparticle content in the polyimide matrix, failure mode was converted from adhesion failure to cohesion one.