Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 773
Filter
1.
J Virol Methods ; 330: 115039, 2024 Sep 30.
Article in English | MEDLINE | ID: mdl-39357745

ABSTRACT

PURPOSE: To propose an efficient, reproducible, and consistent transgenic technology based on plate centrifugation, which is particularly useful for polyethylenimine (PEI) transfection and lentiviral infection. METHODS: We optimized multiple factors that could contribute to transfection efficiency, such as the dosage of the PEI or DNA, the working solution buffer used for diluting the PEI or DNA, the incubation time for the PEI/DNA complexes, and the transfection time. RESULTS: Plate centrifugation led to a 5.46-fold increase in the transfection efficiency of PEI-based transfection while maintaining the cell survival rate. Moreover, the average copy number of viral genes in each genome increased 4.96-fold with plate centrifugation. Plate centrifugation alters the spatial arrangement of the PEI/DNA complexes or lentiviruses, increasing the chances of these complexes or viruses coming into contact with target cells, ultimately resulting in improved transfection or infection efficiency. CONCLUSIONS: We present a protocol based on plate centrifugation for transfection or lentiviral infection that is suitable for genetic modification of primary cells or stem cells.

2.
J Chromatogr A ; 1736: 465422, 2024 Oct 06.
Article in English | MEDLINE | ID: mdl-39383622

ABSTRACT

The characterization of plant extracts is usually accomplished by reverse-phase liquid chromatography, but the development of new complementary approaches, such as HILIC, offers an orthogonal method. In this study, five HILIC stationary phases were evaluated to assess their ability to retain polyphenols. They were selected to cover the main different HILIC mechanisms: bare silica; silica with ethylene bridge; neutral amide; amino; zwitterionic. A total of 31 polyphenol standards were used for the screening, including 9 stilbenes, 8 flavonoids, 6 anthocyanins, and 8 phenolic acids. Three different detections were tested: diode array detector, charged aerosol detector and mass spectrometry. Results indicated that silica supports were not suitable for retaining polyphenols, with no or low retention observed except for anthocyanins. The effectiveness of stationary phases in retention of phenolics following the order related to increased retention: zwitterionic, amide, and amino. The choice of mobile phase also influenced retention. Mobile phases containing TFA as pH modifier limited retention, while formic acid was found to be more effective for polyphenol retention. Ammonium buffers also improved retention but often compromised peak shape. pH changes mainly impacted ionizable compounds, such as phenolic acids, by increasing their retention when they were ionized. DAD was wellsuited for detecting polyphenols that possess aromatic rings, though peak wavelengths depend on the structures of the polyphenols. CAD, while less sensitive than DAD and MS, provided an almost similar response for structurally related compounds, even with gradient elution. MS was the preferred detector for quantification when resolution between compounds was challenging, as it is often the case with natural extracts. The study successfully demonstrated that best HILIC conditions were obtained using an amino stationary phase composed of a polyethylenimine and formic acid-based mobile phase. These conditions were successfully applied to the analysis of stilbenoid-rich extracts from different parts of the vine. The elution order of stilbenoids followed the degree of polymerization. With CAD, the chromatographic profile was more representative of sample composition. It was demonstrated for the first time the interest of a combination of HILIC and CAD for analyzing stilbenes, offering a complementary approach to the classic RP analysis.

3.
ACS Appl Mater Interfaces ; 16(40): 54038-54048, 2024 Oct 09.
Article in English | MEDLINE | ID: mdl-39347984

ABSTRACT

Preparing stable n-type flexible single-walled carbon nanotube (SWCNT)-based thermoelectric films with high thermoelectric (TE) performance is desirable for self-powering wearable electronics but remains a challenge. Here, the interface regulation and thermoelectric enhancement mechanism of ferrocene derivatives on polyethylenimine/single-walled carbon nanotube (PEI/SWCNT) composite films have been explored by doping ferrocene derivatives (f-Fc-OH) into PEI/SWCNT films. The results show that the introduction of f-Fc-OH leads to the formation of "thorn" structures on the surfaces of SWCNT bundles via hydrophilic and hydrophobic interactions, the generated energy-filtering effect improves the thermoelectric properties of the PEI/SWCNT film, and the f-Fc-OH-doped PEI/SWCNT (f-Fc-OH/PEI/SWCNT) achieves the highest room-temperature power factor of 182.22 ± 8.60 µW m-1 K-2 with a Seebeck coefficient of -64.28 ± 0.96 µV K-1 and the corresponding ZT value of 4.69 × 10-3. The Seebeck coefficient retention ratio of the f-Fc-OH/PEI/SWCNT nearly remained 68% after being exposed to air for 3672 h, while the PEI/SWCNT film changed from n-type to p-type after being exposed to air for about 432 h. In addition, the temperature-dependent thermoelectric properties show that the f-Fc-OH/PEI/SWCNT achieves a high power factor of 334.57 µW m-1 K-2 at 353 K. Finally, a flexible TE module consisting of seven pairs of p-n junctions is assembled using the optimum composite film, which produces an open-circuit voltage of 42 mV and a maximum output power of 4.32 µW at a temperature gradient of 60 K. Therefore, this work provides guidance for preparing stable n-type SWCNT-based composite films with enhanced thermoelectric properties, which have potential applications in flexible generators and wearable electronic devices.

4.
Chem Asian J ; : e202400828, 2024 Sep 04.
Article in English | MEDLINE | ID: mdl-39231000

ABSTRACT

Aqueous zinc-ion batteries (AZIBs) are a potential new technology in energy storage due to their high energy density, affordability, and environmental friendliness. Unchecked zinc dendrite formation during cycling still hinders the development of AZIBs, resulting in an unstable interface, a short cycling life, a considerable capacity decline, and security issues. Herein, we demonstrate a novel nanofiber membrane based on a polyethylenimine-polyacrylonitrile (PEI-PAN) polymer produced by electrospinning with entangled nanofibers for AZIBs applications. The as-fabricated PEI/PAN membrane has a porous structure that is homogeneous, tortuous, and linked, with high porosity and superior electrolyte wettability. The PEI/PAN membrane has good thermal stability at 200 °C and high ionic conductivity of up to 5.3 x 10-4 S cm-1. This membrane provides Zn/Zn symmetric cells with an ultralong cycle life of over 250 hours at 3 mA cm-2. Additionally, MnO2/Zn cells outperforms commercial filter paper in terms of cycle stability and rate performance. This work demonstrates a simple technique for fabricating advanced nanofiber membranes for AZIBs to modify Zn2+ deposition behavior and improve Zn dendrite resistance.

5.
Sci Rep ; 14(1): 20564, 2024 09 04.
Article in English | MEDLINE | ID: mdl-39232139

ABSTRACT

High molecular weight polyethylenimine (HMW PEI; branched 25 kDa PEI) has been widely investigated for gene delivery due to its high transfection efficiency. However, the toxicity and lack of targeting to specific cells have limited its clinical application. In the present investigation, L-3, 4-dihydroxyphenylalanine (L-DOPA) was conjugated on HMW PEI in order to target L-type amino acid transporter 1 (LAT-1) and modulate positive charge density on the surface of polymer/plasmid complexes (polyplexes). The results of biophysical characterization revealed that the PEI conjugates are able to form nanoparticles ≤ 180 nm with the zeta potential ranging from + 9.5-12.4 mV. These polyplexes could condense plasmid DNA and protect it against nuclease digestion at the carrier to plasmid ratios higher than 4. L-DOPA conjugated PEI derivatives were complexed with a plasmid encoding human interleukin-12 (hIL-12). Targeted polyplexes showed up to 2.5 fold higher transfection efficiency in 4T1 murine mammary cancer cell line, which expresses LAT-1, than 25 kDa PEI polyplexes prepared in the same manner. The cytotoxicity of these polyplexes was also substantially lower than the unmodified parent HMW PEI. These results support the use of L-3, 4-dihydroxyphenylalanine derivatives of PEI in any attempt to develop a LAT-1 targeted gene carrier.


Subject(s)
Molecular Weight , Plasmids , Polyethyleneimine , Polyethyleneimine/chemistry , Plasmids/genetics , Plasmids/chemistry , Animals , Mice , Cell Line, Tumor , Humans , Dihydroxyphenylalanine/chemistry , Transfection/methods , Gene Transfer Techniques , Interleukin-12/metabolism , Interleukin-12/genetics , Large Neutral Amino Acid-Transporter 1/metabolism , Large Neutral Amino Acid-Transporter 1/genetics , Nanoparticles/chemistry , DNA/chemistry
6.
Adv Healthc Mater ; : e2402688, 2024 Sep 11.
Article in English | MEDLINE | ID: mdl-39258393

ABSTRACT

Antigen delivery via respiratory mucosal surfaces is an interesting needle-free option for vaccination. Nonetheless, it demands for the design of especially tailored formulations. Here, lipid/poly(lactic-co-glycolic) acid (PLGA) hybrid nanoparticles (hNPs) for the combined delivery of an antigen, ovalbumin (Ova), and an adjuvant, synthetic unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) motifs, is developed. A panel of Ova/CpG-loaded lipid@PLGA hNPs with tunable size and surface is attained by exploiting two lipid moieties, 1,2 distearoil-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) and monophosphoryl lipid A (MPLA), with or without polyethyleneimine (PEI). It is gained insights on the lipid@PLGA hNPs through a combination of techniques to analytically determine the specific moiety on the surface, the spatial distribution of the components and the internal structure of the nanoplatforms. The collected results suggest that PEI plays a role of paramount importance not only in promoting in vitro antigen escape from lysosomes and enhancing antigen cross-presentation, but also in determining the arrangement of the moieties in the final architecture of the hNPs. Though multicomponent PEI-engineered lipid@PLGA hNPs turn out as a viable strategy for delivery of antigens and adjuvant to the respiratory mucosa, tunable nanoparticle features are achievable only through the optimal selection of the components and their relative amounts.

7.
ACS Appl Mater Interfaces ; 16(34): 44376-44385, 2024 Aug 28.
Article in English | MEDLINE | ID: mdl-39145762

ABSTRACT

Gene transfection, defined by the delivery of nucleic acids into cellular compartments, stands as a crucial procedure in gene therapy. While branched polyethylenimine (PEI) is widely regarded as the "gold standard" for nonviral vectors, its cationic nature presents several issues, including nonspecific protein adsorption and notable cytotoxicity. Additionally, it often fails to achieve high transfection efficiency, particularly with hard-to-transfect cell types. To overcome these challenges associated with PEI as a vector for plasmid DNA (pDNA), the photothermal agent indocyanine green (ICG) is integrated with PEI and pDNA to form the PEI/ICG/pDNA (PI/pDNA) complex for more efficient and safer gene transfection. The negatively charged ICG serves a dual purpose: neutralizing PEI's excessive positive charges to reduce cytotoxicity and, under near-infrared irradiation, inducing local heating that enhances cell membrane permeability, thus facilitating the uptake of PI/pDNA complexes to boost transfection efficiency. Using pDNA encoding vascular endothelial growth factor as a model, our system shows enhanced transfection efficiency in vitro for hard-to-transfect endothelial cells, leading to improved cell proliferation and migration. Furthermore, in vivo studies reveal the therapeutic potential of this system in accelerating the healing of infected wounds by promoting angiogenesis and reducing inflammation. This approach offers a straightforward and effective method for gene transfection, showing potentials for tissue engineering and cell-based therapies.


Subject(s)
Indocyanine Green , Plasmids , Polyethyleneimine , Transfection , Indocyanine Green/chemistry , Indocyanine Green/pharmacology , Polyethyleneimine/chemistry , Humans , Transfection/methods , Animals , Plasmids/genetics , Plasmids/metabolism , Plasmids/chemistry , Mice , DNA/chemistry , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/genetics , Human Umbilical Vein Endothelial Cells , Cell Proliferation/drug effects
8.
Small Methods ; : e2400643, 2024 Aug 19.
Article in English | MEDLINE | ID: mdl-39161085

ABSTRACT

Additive engineering has emerged as a promising strategy to address the inherent instability challenges of perovskite solar cells (PSCs) in the pursuit of commercial viability. However, achieving multifunctionality using a singular additive remains a considerable challenge. In this study, a novel comb-like multifunctional perfluoroalkyl-g-polyethylenimmonium iodide (FPEI·HI) as additives to the PbI2 precursor solution to facilitate the formation of high-quality and water-resistant perovskite films is presented. FPEI·HI establishes robust interactions with both formamidinium iodide (FAI) and PbI2, mediated by hydrogen bonding and Lewis acid-base interactions. These interactions play a pivotal role in simultaneously passivating negative and positive charged defects within the perovskite structure. Furthermore, the inclusion of perfluoroalkyl chains serves as resilience against moisture intrusion. As a consequence of these effects, a notably high device efficiency of 24.29% is achieved, demonstrating comprehensive improvement in various photovoltaic parameters compared to the control device (22.51%). Notably, unencapsulated devices exhibit remarkable stability in high-humidity environments, retaining 90% of their initial efficiency even after 2500 h of storage. This work underscores the efficacy of FPEI·HI as a critical enabler for enhancing the stability and efficiency of perovskite solar cells, marking a significant stride toward their commercialization.

9.
Biol Pharm Bull ; 47(7): 1396-1404, 2024.
Article in English | MEDLINE | ID: mdl-39085138

ABSTRACT

Estimation of the continuous hemodiafiltration (CHDF) clearance (CLCHDF) of ganciclovir (GCV) is crucial for achieving efficient treatment outcomes. Here, we aimed to clarify the contribution of diafiltration, adsorption, and hematocrit level to the CLCHDF of GCV in an in vitro CHDF model using three membranes: polyacrylonitrile and sodium methallyl sulfonate copolymer coated with polyethylenimine (AN69ST); polymethylmethacrylate (PMMA); and polysulfone (PS). In vitro CHDF was performed with effluent flow rates (Qe) of 800, 1500, and 3000 mL/h. The initial GCV concentration was 10 µg/mL while that of human serum albumin (HSA) was 0 or 5 g/dL. The CLCHDF, diafiltration rates, and adsorption rates were calculated. The whole blood-to-plasma ratio (R) of GCV for a hematocrit of 0.1 to 0.5 was determined using blood samples with 0.5 to 100 µg/mL of GCV. The in vitro CHDF experiment using AN69ST, PMMA, and PS membranes showed that the total CLCHDF values were almost the same as the Qe and not influenced by the HSA concentration. The diafiltration rate exceeded 88.1 ± 2.8% while the adsorption rate was lower than 9.4 ± 9.4% in all conditions. The R value was 1.89 ± 0.11 and was similar at all hematocrit levels and GCV concentrations. In conclusion, diafiltration mainly contributes to the CLCHDF of GCV, rather than adsorption. Hematocrit levels might not affect the relationship between the plasma and blood CLCHDF of GCV, and the CLCHDF of GCV can be estimated from the Qe and R, at least in vitro.


Subject(s)
Acrylic Resins , Ganciclovir , Hemodiafiltration , Humans , Hemodiafiltration/methods , Adsorption , Ganciclovir/pharmacokinetics , Ganciclovir/blood , Ganciclovir/administration & dosage , Hematocrit , Acrylic Resins/chemistry , Antiviral Agents/blood , Antiviral Agents/pharmacokinetics , Polymethyl Methacrylate/chemistry , Polymers/chemistry , Membranes, Artificial
10.
Small Methods ; : e2400345, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38966877

ABSTRACT

Ultraviolet (UV) light is typically needed to activate inverted organic photovoltaics (OPVs) with zinc oxide (ZnO) as electron transporting layer (ETL) for higher efficiency. However, UV light is a major cause for the degradation of organic active layers in OPVs. This is a contradiction that UV light activation enhances the efficiency but UV illumination deteriorates the stability. It is important to solve this contradiction to develop UV light activation-free OPV devices. Herein, a method of aqueous polyethylenimine ethoxylated (PEIE) soaking on ZnO is reported to realize UV light activation-free OPV devices. The S-shape in current density-voltage (J-V) characteristics of devices tested without UV light activation is eliminated through the treatment of aqueous PEIE soaking on ZnO. The treatment reduces the oxygen adsorbates, which is confirmed by Kelvin probe and X-ray photoelectron spectroscopy. A 10.08 cm2 organic photovoltaic module with the treated ZnO as ETL showed high photovoltaic performance: VOC = 5.68 V, JSC = 2.7 mA cm-2, FF = 75.1%, and POutput = 11.5 mW cm-2 tested with the UV filter (light intensity of 0.788 sun). UV light activation is not needed for the modules to obtain high efficiency.

11.
J Sep Sci ; 47(13): e2400154, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38948935

ABSTRACT

Glycosylation and phosphorylation rank as paramount post-translational modifications, and their analysis heavily relies on enrichment techniques. In this work, a facile approach was developed for the one-step simultaneous enrichment and stepwise elution of glycoproteins and phosphoproteins. The core of this approach was the application of the novel titanium (IV) ion immobilized poly(glycidyl methacrylate) microparticles functionalized with dendrimer polyethylenimine and phytic acid. The microparticles possessed dual enrichment capabilities due to their abundant titanium ions and hydroxyl groups on the surface. They demonstrate rapid adsorption equilibrium (within 30 min) and exceptional adsorption capacity for ß-casein (1107.7 mg/g) and horseradish peroxidase (438.6 mg/g), surpassing that of bovine serum albumin (91.7 mg/g). Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was conducted to validate the enrichment capability. Experimental results across various biological samples, including standard protein mixtures, non-fat milk, and human serum, demonstrated the remarkable ability of these microparticles to enrich low-abundance glycoproteins and phosphoproteins from biological samples.


Subject(s)
Dendrimers , Glycoproteins , Phosphoproteins , Polyethyleneimine , Polymethacrylic Acids , Titanium , Glycoproteins/chemistry , Phosphoproteins/chemistry , Polyethyleneimine/chemistry , Dendrimers/chemistry , Humans , Titanium/chemistry , Polymethacrylic Acids/chemistry , Hydrophobic and Hydrophilic Interactions , Surface Properties , Animals , Particle Size , Adsorption , Cattle
12.
Sci Rep ; 14(1): 15667, 2024 07 08.
Article in English | MEDLINE | ID: mdl-38977741

ABSTRACT

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6's large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde's spacing arm, reducing spatial hindrance and enhancing enzyme-substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.


Subject(s)
Acrylamide , Dopamine , Enzymes, Immobilized , Polyethyleneimine , Serum Albumin, Bovine , Trypsin , Polyethyleneimine/chemistry , Dopamine/chemistry , Dopamine/metabolism , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Acrylamide/chemistry , Trypsin/chemistry , Trypsin/metabolism , Animals , Cattle , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/metabolism , Porosity , Hydrophobic and Hydrophilic Interactions , Hemoglobins/chemistry , Hemoglobins/metabolism , Proteolysis
13.
Colloids Surf B Biointerfaces ; 241: 114052, 2024 Sep.
Article in English | MEDLINE | ID: mdl-38917667

ABSTRACT

Due to their resemblance to the fibrillar structure of the extracellular matrix, electrospun nanofibrous meshes are currently used as porous and mechanically stable scaffolds for cell culture. In this study, we propose an innovative methodology for growing peptide sequences directly onto the surface of electrospun nanofibers. To achieve this, electrospun fibers were produced from a poly(acrylic acid)/poly(vinyl alcohol) blend that was thermally crosslinked and subjected to a covalent coating of branched poly(ethylenimine). The exposed amino functionalities on the fiber surface were then used for the direct solid-phase synthesis of the RGD peptide sequence. In contrast to established strategies, mainly involving the grafting of pre-synthesized peptides onto the polymer chains before electrospinning or onto the nanofibers surface, this method allows for the concurrent synthesis and anchoring of peptides to the substrate, with potential applications in combinatorial chemistry. The incorporation of this integrin-binding motive significantly enhanced the nanofibers' ability to capture human cervical carcinoma (HeLa) cells, selected as a proof of concept to assess the functionalities of the developed material.


Subject(s)
Acrylic Resins , Nanofibers , Polyethyleneimine , Polyvinyl Alcohol , Humans , Polyvinyl Alcohol/chemistry , Acrylic Resins/chemistry , Nanofibers/chemistry , HeLa Cells , Polyethyleneimine/chemistry , Tissue Scaffolds/chemistry , Peptides/chemistry , Oligopeptides/chemistry , Surface Properties
14.
Materials (Basel) ; 17(12)2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38930282

ABSTRACT

Amines supported on porous solid materials have a high CO2 adsorption capacity and low regeneration temperature. However, the high amine load on such substrates and the substrate itself may lead to substantial pressure drop across the reactor. Herein, we compare the CO2 adsorption capacity and pressure drop of fumed silica powder to 3D-printed monolithic fumed silica structures, both functionalized by polyethylenimine (PEI), and find a drastically reduced pressure drop for 3D-printed substrates (0.01 bar vs. 0.76 bar) in the sorption bed with equal CO2 adsorption capacity. Furthermore, the effect of 3D-printing nozzle diameter and PEI loading on the adsorption capacity are investigated and the highest capacities (2.0 mmol/g at 25 °C with 5000 ppm CO2) are achieved with 0.4 mm nozzle size and 34 wt% PEI loading. These high capacities are achieved since the 3D printing and subsequent sintering (700 °C) of monolithic samples does not compromise the surface area of the fumed silica. Finally, the comparison between 3D-printed monoliths and extruded granulate of varying diameter reveals that the ordered channel system of 3D-printed structures is superior to randomly oriented granulate in terms of CO2 adsorption capacity.

15.
Methods Mol Biol ; 2810: 75-83, 2024.
Article in English | MEDLINE | ID: mdl-38926273

ABSTRACT

Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.


Subject(s)
Bioreactors , Recombinant Proteins , Transfection , Transfection/methods , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Humans , Animals , Cell Line , Cell Culture Techniques/methods , Gene Expression
16.
Methods Mol Biol ; 2810: 99-121, 2024.
Article in English | MEDLINE | ID: mdl-38926275

ABSTRACT

The continuous improvement of expression platforms is necessary to respond to the increasing demand for recombinant proteins that are required to carry out structural or functional studies as well as for their characterization as biotherapeutics. While transient gene expression (TGE) in mammalian cells constitutes a rapid and well-established approach, non-clonal stably transfected cells, or "pools," represent another option, which is especially attractive when recurring productions of the same protein are required. From a culture volume of just a few liters, stable pools can provide hundreds of milligrams to gram quantities of high-quality secreted recombinant proteins.In this chapter, we describe a highly efficient and cost-effective procedure for the generation of Chinese Hamster Ovary cell stable pools expressing secreted recombinant proteins using commercially available serum-free media and polyethylenimine (PEI) as the transfection reagent. As a specific example of how this protocol can be applied, the production and downstream purification of recombinant His-tagged trimeric SARS-CoV-2 spike protein ectodomain (SmT1) are described.


Subject(s)
Cricetulus , Polyethyleneimine , Recombinant Proteins , Spike Glycoprotein, Coronavirus , Transfection , CHO Cells , Animals , Recombinant Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection/methods , Polyethyleneimine/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/biosynthesis , Spike Glycoprotein, Coronavirus/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Cricetinae , Culture Media, Serum-Free
17.
Gels ; 10(6)2024 May 23.
Article in English | MEDLINE | ID: mdl-38920907

ABSTRACT

This work studied the low-temperature sorption of carbon dioxide on impregnated silica gel. An impregnating agent was used polyethyleneimine. The content of the impregnating agent in the silica gel matrix was 33.4 wt.%. Material properties such as the Brunauer-Emmett-Teller (BET) surface area, pore distribution, total pore volume, and thermal stability of the impregnated material were determined for the sample. During the measurement of the adsorption-desorption cycles, the loss of the impregnating agent in the material matrix was also determined. Due to the decrease in the content of polyethyleneimine, the sorption capacity of the adsorbent for CO2 also decreased. It was found that after the 20th adsorption-desorption cycle, the content of the impregnating agent in the adsorbent dropped by 3.15 wt.%, and, as a result, the adsorption capacity for CO2 dropped to almost half.

18.
Iran J Basic Med Sci ; 27(8): 952-958, 2024.
Article in English | MEDLINE | ID: mdl-38911238

ABSTRACT

Objectives: For safe and effective gene therapy, the ability to deliver the therapeutic nucleic acid to the target sites is crucial. In this study, lactosylated lipid phosphate calcium nanoparticles (lac-LCP) were developed for targeted delivery of pDNA to the hepatocyte cells. The lac-LCP formulation contained lactose-modified cholesterol (CHL), a ligand that binds to the asialoglycoprotein receptor (ASGR) expressed on hepatocytes, and polyethyleneimine (PEI) in the core. Materials and Methods: Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) were used to monitor the chemical modification, and the physicochemical properties of NPs were studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). To evaluate transfection efficiency, cellular uptake and GFP expression were assessed using fluorescence microscopy and flow cytometry. Results: The results revealed that lactose-targeted particles (lac-LCP) had a significant increase in cellular uptake by hepatocytes. The inclusion of a low molecular weight PEI (1.8 KDa) with a low PEI/pDNA ratio of 1 in the core of LCP, elicited high degrees of GFP protein expression (by 5 and 6-fold), which exhibited significantly higher efficiency than PEI 1.8 KDa and Lipofectamine. Conclusion: The successful functionalization and nuclear delivery of LCP NPs described here indicate its promise as an efficient delivery vector to hepatocyte nuclei.

19.
ACS Appl Mater Interfaces ; 16(24): 31098-31113, 2024 Jun 19.
Article in English | MEDLINE | ID: mdl-38845418

ABSTRACT

Cotton-based textiles are ubiquitous in daily life and are prime candidates for application in wearable triboelectric nanogenerators. However, pristine cotton is vulnerable to bacterial attack, lacks antioxidant and ultraviolet (UV)-protective abilities, and shows lower triboelectric charge generation against tribonegative materials because it is present in the neutral region of the triboelectric series. To overcome such drawbacks, herein, a facile layer-by-layer method is proposed, involving the deposition of alternate layers of polyethylenimine (PEI) and sodium alginate (SA) on cotton. Such modified fabric remains breathable and flexible, retains its comfort properties, and simultaneously shows multifunctionalities and improved triboelectric output, which are retained even after 50 home laundering cycles. Also, the modified fabric becomes more tribopositive than nylon, silk, and wool. A triboelectric nanogenerator consisting of modified cotton and polyester fabric is proposed that shows a maximum power density of 338 mW/m2. An open-circuit voltage of ∼97.3 V and a short-circuit current of ∼4.59 µA are obtained under 20 N force and 1 Hz tapping frequency. Further, the modified cotton exhibits excellent antibacterial, antioxidant, and UV-protective properties because of the incorporation of PEI, and its moisture management properties are retained due to the presence of sodium alginate in the layer. This study provides a simple yet effective approach to obtaining durable multifunctionalities and improved triboelectric performance in cotton substrates.

20.
ChemMedChem ; 19(15): e202400011, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38740551

ABSTRACT

The innate immune system is an evolutionarily conserved pathogen recognition mechanism that serves as the first line of defense against tissue damage or pathogen invasion. Unlike the adaptive immunity that recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs. Thus low-molecular weight (600 Da) BPEI may be able to counter dysregulated inflammation in chronic wounds and promote successful repair following tissue injury.


Subject(s)
Cytokines , Escherichia coli , Klebsiella pneumoniae , Lipopolysaccharides , Monocytes , Polyethyleneimine , Humans , Cytokines/metabolism , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/immunology , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Lipopolysaccharides/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Escherichia coli/drug effects , Molecular Weight , Pathogen-Associated Molecular Pattern Molecules/metabolism , Pathogen-Associated Molecular Pattern Molecules/immunology
SELECTION OF CITATIONS
SEARCH DETAIL