-
Nanomaterials (Basel, Switzerland) Nov 2023Nanoparticles are increasingly being used by industry to enhance the outcomes of various chemical processes. In many cases, these processes involve over-dosages that...
Nanoparticles are increasingly being used by industry to enhance the outcomes of various chemical processes. In many cases, these processes involve over-dosages that compensate for particle losses. At best, these unique waste streams end up in landfills. This circumstance is inefficient and coupled with uncertain impacts on the environment. Pozzolanic nanoparticle treatments have been found to provide remarkable benefits for strength restoration and the mitigation of durability problems in ordinary Portland cement and concrete. These treatments have been accompanied by significant particle losses stemming from over-dosages and instability of the colloidal suspensions that are used to deliver these materials into the pore structure. In this study, new methods involving simple tools have been developed to monitor and sustain suspension stability. Turbidity measurement was introduced to monitor the progress of electrokinetic nanoparticle treatment. This tool made it possible to amend a given dosing strategy in real time while it remains possible to make effective treatment adjustments. By monitoring the particle stability and using pH and electric field controls to avoid suspension collapse, successful electrokinetic treatment dosage strategies were demonstrated using 20 nm NALCO 1056 alumina-coated silica particles. These trials indicated that turbidity measurements could track the visually imperceptible phenomena of particle flocking early on at the inception of its development. Suspensions of these nanoparticles were successfully delivered into 5 cm diameter by 10 cm tall hardended cement paste (HCP) specimens by monitoring fluid turbidity along with the specific gravity and using these values to guide the active management of the treatment dosage and pH. Under this new strategy, these losses were reduced to 5% as compared to the 80% losses associated with other treatment approaches. The relationship between the turbidity and the specific gravity was found to be linear. These plots indicated regions of turbidity and specific gravity that were associated with particle flocking. The tools, guidelines, and strategies developed in this work made it possible to manage efficient (low-particle-loss) electrokinetic nanoparticle treatments by signaling in real time when adjustments to electric field, pH, and particle dosage increments were needed.
PubMed: 38063741
DOI: 10.3390/nano13233045 -
Experimental Eye Research Aug 2023The single-cell RNA-sequencing (scRNA-seq) technique is used to explore the biological characteristics of tissues under pathological and physiological conditions that...
The single-cell RNA-sequencing (scRNA-seq) technique is used to explore the biological characteristics of tissues under pathological and physiological conditions that include certain chronic eye diseases. Harvesting of single-cell suspensions is one challenge inherent to scRNA-seq procedures. This study aimed to use an optimized method to digest a whole mouse cornea to harvest single-cell suspensions. We utilized five different mouse cornea digestion methods to obtain single-cell suspensions: (1) 5 dissected mouse corneas were cut into pieces (∼0.5 mm) and digested in trypsin for 10 min, and this digestion was repeated for 10 cycles; (2) 5 dissected mouse corneas were cut into pieces and incubated with 5 mg/ml collagenase A at 37 °C for 1h and then further digested in trypsin at 37 °C for 10 min; (3) used the same approach as that used in method 2, but the second digestion step was performed in TrypLE for 20 min; (4) used the same approach as that used in method 2, but the concentration of collagenase A was 2 mg/ml and the incubation time was 2h; (5) used the same approach as that used in method 3, but the corneas were incubated in 2 mg/ml collagenase A at 37 °C for 2h. Trypan blue staining was used to calculate the cell viability and agglomeration rate. The cell types and percentages were determined using immunofluorescence staining. RNA integrity number (RIN) was measured by Agilent 2100. Method 1 showed the lowest cell yield (0.375 × 10), epithelial cell percentage, and less than 70% cell viability, thus not a proper protocol. Method 2 showed the highest cell viability (over 90%), percentage of single-cell (89.53%), and high cell quantity (1.05 × 10). Method 3 had a significantly lower cell viability (55.30%). Cell agglomeration rates of method 4 and 5 reached up to 20% and 13%, and with lower cell viability (72.51%, 59.87%, respectively) and decreased epithelial cell rate compared to method 2 and 3. The results suggest that method 2 (5 mg/ml collagenase A and trypsin) is a preferred protocol for digesting mouse cornea to obtain single-cell suspension which achieves the criterion of single-cell RNA sequencing.
Topics: Mice; Animals; Trypsin; Suspensions; Collagenases; Cornea; RNA
PubMed: 37336469
DOI: 10.1016/j.exer.2023.109544 -
International Journal of Biological... Dec 2023In this work, magnetic flocculant (FeO@PP) was synthesized using plant polyphenol (PP) as a shaping ligand via in situ self-assembly. Characterization results revealed...
In this work, magnetic flocculant (FeO@PP) was synthesized using plant polyphenol (PP) as a shaping ligand via in situ self-assembly. Characterization results revealed that FeO@PP exhibited uniform particle size and excellent dispersibility with PP coating amount of 16.4 %. Experimental results suggested that FeO@PP showed excellent turbidity removal efficiency in a wide pH range (3.0-10) and initial turbidity range (50-2000 NTU). Under the optimal conditions, FeO@PP achieved 95.2 % of turbidity removal for simulated kaolin suspension and 96.9 % for actual wastewater. FeO@PP exhibited excellent recycling and reusability properties, with high recycling efficiency of 93.3 % even after the fifth cycle. Microscopic observation revealed the formation process of magnetic flocs, involving particle aggregation, chain and cluster formation, and dense network aggregate formation. The structural characteristics and size of magnetic flocs were found to be significantly influenced by the combined effects of magnetic force, electric charge, van der Waals force, and functional groups on the surface of PP. The extended Deryaguin-Landau-Verwey-Overbeek models indicated that magnetic interactions were the primary mechanism for magnetic flocculation, accompanied by charge neutralization, adsorption bridging, sweeping, and net trapping.
Topics: Water Purification; Kaolin; Flocculation; Polyphenols; Suspensions; Magnetic Phenomena
PubMed: 37652337
DOI: 10.1016/j.ijbiomac.2023.126578 -
Pharmaceuticals (Basel, Switzerland) Sep 2023Non-small-cell lung cancer (NSCLC) mortality and new case rates are both on the rise. Most patients have fewer treatment options accessible due to side effects from...
Non-small-cell lung cancer (NSCLC) mortality and new case rates are both on the rise. Most patients have fewer treatment options accessible due to side effects from drugs and the emergence of drug resistance. Bedaquiline (BQ), a drug licensed by the FDA to treat tuberculosis (TB), has demonstrated highly effective anti-cancer properties in the past. However, it is difficult to transport the biological barriers because of their limited solubility in water. Our study developed a UPLC method whose calibration curves showed linearity in the range of 5 ng/mL to 500 ng/mL. The UPLC method was developed with a retention time of 1.42 and high accuracy and precision. Its LOQ and LOD were observed to be 10 ng/mL and 5 ng/mL, respectively, whereas in the formulation, capmul MCM C10, Poloxamer 188, and PL90G were selected as solid lipids, surfactants, and co-surfactants, respectively, in the development of SLN. To combat NSCLC, we developed solid lipid nanoparticles (SLNs) loaded with BQ, whereas BQ suspension is prepared by the trituration method using acacia powder, hydroxypropyl methylcellulose, polyvinyl acrylic acid, and BQ. The developed and optimized BQ-SLN3 has a particle size of 144 nm and a zeta potential of (-) 16.3 mV. whereas BQ-loaded SLN3 has observed entrapment efficiency (EE) and loading capacity (LC) of 92.05% and 13.33%, respectively. Further, BQ-loaded suspension revealed a particle size of 1180 nm, a PDI of 0.25, and a zeta potential of -0.0668. whereas the EE and LC of BQ-loaded suspension were revealed to be 88.89% and 11.43%, respectively. The BQ-SLN3 exhibited insignificant variation in particle size, homogeneous dispersion, zeta potential, EE, and LC and remained stable over 90 days of storage at 25 °C/60% RH, whereas at 40 °C/75% RH, BQ-SLN3 observed significant variation in the above-mentioned parameters and remained unstable over 90 days of storage. Meanwhile, the BQ suspension at both 25 °C (60% RH) and 40 °C (75% RH) was found to be stable up to 90 days. The optimized BQ-SLN3 and BQ-suspension were in vitro gastrointestinally stable at pH 1.2 and 6.8, respectively. The in vitro drug release of BQ-SLN3 showed 98.19% up to 12 h at pH 7.2 whereas BQ suspensions observed only 40% drug release up to 4 h at pH 7.2 and maximum drug release of >99% within 4 h at pH 4.0. The mathematical modeling of BQ-SLN3 followed first-order release kinetics followed by a non-Fickian diffusion mechanism. After 24 to 72 h, the IC50 value of BQ-SLN3 was 3.46-fold lower than that of the BQ suspension, whereas the blank SLN observed cell viability of 98.01% and an IC50 of 120 g/mL at the end of 72 h. The bioavailability and higher biodistribution of BQ-SLN3 in the lung tumor were also shown to be greater than those of the BQ suspension. The effects of BQ-SLN3 on antioxidant enzymes, including MDA, SOD, CAT, GSH, and GR, in the treated group were significantly improved and reached the level nearest to that of the control group of rats over the cancer group of rats and the BQ suspension-treated group of rats. Moreover, the pharmacodynamic activity resulted in greater tumor volume and tumor weight reduction by BQ-SLN3 over the BQ suspension-treated group. As far as we are aware, this is the first research to look at the potential of SLN as a repurposed oral drug delivery, and the results suggest that BQ-loaded SLN3 is a better approach for NSCLC due to its better action potential.
PubMed: 37765117
DOI: 10.3390/ph16091309 -
Clinical Pharmacology in Drug... May 2024Belumosudil is a selective rho-associated coiled-coil-containing protein kinase 2 inhibitor in clinical use for the treatment of chronic graft-versus-host disease. The... (Randomized Controlled Trial)
Randomized Controlled Trial Comparative Study
Two-Part Phase 1 Study to Evaluate the Taste Profile of Novel Belumosudil Oral Suspensions and Assess the Relative Bioavailability and Food Effect of the Selected Belumosudil Oral Suspension Compared With Oral Tablet Reference in Healthy Male Participants.
Belumosudil is a selective rho-associated coiled-coil-containing protein kinase 2 inhibitor in clinical use for the treatment of chronic graft-versus-host disease. The current tablet formulation may be inappropriate for children or adults with dysphagia and/or upper gastrointestinal manifestations of chronic graft-versus-host disease. This study (NCT04735822) assessed the taste and palatability of oral suspensions of belumosudil, evaluated the relative bioavailability of an oral suspension versus the tablet formulation, and characterized the effect of food on the pharmacokinetics of an oral suspension. Addition of sweetener and/or flavor vehicle improved the taste. Relative bioavailability of 200-mg doses of the oral suspension and tablet in the fed state was similar for belumosudil and its metabolites (KD025m1 and KD025m2), but absorption was faster with the oral suspension (median time to maximum concentration: 2 vs 3 hours). Administration of the oral suspension with food increased exposure compared with fasted administration, with maximum observed concentration being increased by 16% and area under the concentration-time curve from time 0 to the last measurable concentration (AUC) by 19%. Safety and tolerability were consistent with the known safety profile of belumosudil. These results may support administration of a 200-mg belumosudil oral suspension with or without food.
Topics: Humans; Male; Biological Availability; Administration, Oral; Tablets; Adult; Food-Drug Interactions; Taste; Suspensions; Young Adult; Healthy Volunteers; Cross-Over Studies; Area Under Curve; Middle Aged; Double-Blind Method; Protein Kinase Inhibitors
PubMed: 38345529
DOI: 10.1002/cpdd.1378 -
Infection and Immunity Jul 2023Parasitic diseases are a major public health problem worldwide. Plant-derived products appear to be ideal candidates from a biotechnological perspective, being...
Parasitic diseases are a major public health problem worldwide. Plant-derived products appear to be ideal candidates from a biotechnological perspective, being sustainable and environmentally friendly. The antiparasitic properties of have been attributed to some of its components, including papain and other compounds that are concentrated in the latex and seeds. This study demonstrated a high and insignificantly different cysticidal activity of soluble extract that was obtained after the disruption of nontransformed wild-type (WT) cells as well as transformed papaya calluses (PC-9, PC-12, and PC-23) and papaya cell suspensions (CS-9, CS-12, and CS-23). , cell suspensions of CS-WT and CS-23 that had been previously lyophilized were tested with respect to their cysticidal effects, compared with those of three commercial antiparasitic drugs. CS-WT and CS-23 together reduced the number of cysticerci, the number of buds, and the percentage of calcified cysticerci in a similar extent to albendazole and niclosamide, whereas ivermectin was less effective. Mice were then orally immunized with CS-23 that expressed the anti-cysticercal KETc7 antigen (10 μg/mouse), CS-WT (10 mg/mouse), or both together to evaluate their preventive properties. CS-23 and CS-WT significantly reduced the expected parasite and increased the percentage of calcified cysticerci as well as recovery, being more effective when employed together. The results reported in this study support the feasibility of the development of an anti-cysticercosis vaccine from cells of in cultures, as they are a source of an anthelmintic, natural, and reproducible product.
Topics: Mice; Animals; Carica; Suspensions; Albendazole; Plant Extracts; Seeds
PubMed: 37341599
DOI: 10.1128/iai.00517-22 -
Proceedings of the National Academy of... Dec 2023A hallmark of concentrated suspensions is non-Newtonian behavior, whereby the viscosity increases dramatically once a characteristic shear rate or stress is exceeded....
A hallmark of concentrated suspensions is non-Newtonian behavior, whereby the viscosity increases dramatically once a characteristic shear rate or stress is exceeded. Such strong shear thickening is thought to originate from a network of frictional particle-particle contact forces, which forms under sufficiently large stress, evolves dynamically, and adapts to changing loads. While there is much evidence from simulations for the emergence of this network during shear thickening, experimental confirmation has been difficult. Here, we use suspensions of piezoelectric nanoparticles and exploit the strong local stress focusing within the network to activate charge generation. This charging can then be detected in the measured ac conductance and serve as a signature of frictional contact formation. The direct link between stress-activated frictional particle interactions and piezoelectric suspension response is further demonstrated by tracking the emergence of structural memory in the contact network under oscillatory shear and by showing how stress-activated friction can drive mechano-transduction of chemical reactions with nonlinear reaction kinetics. Taken together, this makes the ac conductance of piezoelectric suspensions a sensitive in-situ reporter of the micromechanics associated with frictional interactions.
PubMed: 38015840
DOI: 10.1073/pnas.2310088120 -
Protein Expression and Purification Oct 2023The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it...
A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: Yield improvements, bioreactor production and downstream processing.
The human cell line HEK293 is one of the preferred choices for manufacturing therapeutic proteins and viral vectors for human applications. Despite its increased use, it is still considered in disadvantage in production aspects compared to cell lines such as the CHO cell line. We provide here a simple workflow for the rapid generation of stably transfected HEK293 cells expressing an engineered variant of the SARS-CoV-2 Receptor Binding Domain (RBD) carrying a coupling domain for linkage to VLPs through a bacterial transpeptidase-sortase (SrtA). To generate stable suspension cells expressing the RBD-SrtA, a single two plasmids transfection was performed, with hygromycin selection. The suspension HEK293 were grown in adherent conditions, with 20% FBS supplementation. These transfection conditions increased cell survival, allowing the selection of stable cell pools, which was otherwise not possible with standard procedures in suspension. Six pools were isolated, expanded and successfully re-adapted to suspension with a gradual increase of serum-free media and agitation. The complete process lasted four weeks. Stable expression with viability over 98% was verified for over two months in culture, with cell passages every 4-5 days. With process intensification, RBD-SrtA yields reached 6.4 μg/mL and 13.4 μg/mL in fed-batch and perfusion-like cultures, respectively. RBD-SrtA was further produced in fed-batch stirred tank 1L-bioreactors, reaching 10-fold higher yields than perfusion flasks. The trimeric antigen displayed the conformational structure and functionality expected. This work provides a series of steps for stable cell pool development using suspension HEK293 cells aimed at the scalable production of recombinant proteins.
Topics: Humans; HEK293 Cells; COVID-19; SARS-CoV-2; Bioreactors; Recombinant Proteins
PubMed: 37201590
DOI: 10.1016/j.pep.2023.106295 -
Scientific Reports Aug 2023We study the rheological behaviour of bidisperse suspensions in three dimensions under a non-uniform shear flow, made by the superimposition of a linear shear and a...
We study the rheological behaviour of bidisperse suspensions in three dimensions under a non-uniform shear flow, made by the superimposition of a linear shear and a sinusoidal disturbance. Our results show that (i) only a streamwise disturbance in the shear-plane alters the suspension dynamics by substantially reducing the relative viscosity, (ii) with the amplitude of the disturbance determining a threshold value for the effect to kick-in and its wavenumber controlling the amount of reduction and which of the two phases is affected. We show that, (iii) the rheological changes are caused by the effective separation of the two phases, with the large or small particles layering in separate regions. We provide a physical explanation of the phase separation process and of the conditions necessary to trigger it. We test the results in the whole flow curve, and we show that the mechanism remains substantially unaltered, with the only difference being the nature of the interactions between particles modified by the phase separation.
PubMed: 37652962
DOI: 10.1038/s41598-023-41587-3 -
Plant Methods Oct 2023This study aims to establish cell suspension culture, its maintenance and induction of somatic embryogenesis, and in vitro plant regeneration in Cenchrus ciliaris L....
BACKGROUND
This study aims to establish cell suspension culture, its maintenance and induction of somatic embryogenesis, and in vitro plant regeneration in Cenchrus ciliaris L. Suspension cultures are relatively homogenous cell lines facilitating uniform access to nutrition. These are ideal sources of competent cells for genetic transformation.
RESULTS
Callus was initiated by culturing immature inflorescences of Cenchrus ciliaris cv. IGFRI-3108 on Murashige and Skoog (MS) medium containing 3 mg l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l 6-benzylaminopurine (BAP) with 30 g l sucrose. Cell suspension cultures were established in liquid MS medium using an inoculum size of 10 g l. These were maintained to achieve embryogenic cell/regeneration competent cultures. Growth curve analysis and a subculture interval of 20 days were determined to harvest cells at the end of the exponential phase. The cell doubling time was found to be 11 days. Somatic embryogenesis was accomplished in MS medium containing 1 mg l 2,4-D, 1 mg l BAP along with growth adjuvants, 300 mg l casein hydrolysate, 400 mg l glutamine and 300 mg l proline. The highest number (16 ± 3.78/per inoculum) of shoots regenerated on this medium. The elongation and rooting of shoots were recorded on basal MS and ½ MS media, respectively. Rooted plants were successfully transferred to pots containing a Soilrite and cocopeat mixture in a 3:1 proportion for 3-4 weeks and later successfully acclimatized in the greenhouse with a 60% survival rate. The genetic fidelity of 12 regenerated plants was analysed using RAPD primers that were genetically identical to the mother plant.
CONCLUSION
Cell suspension culture-based in vitro plant regeneration of C. ciliaris involved the establishment, maintenance and progression of somatic embryogenesis during shoot and root development. The inherent limitation of callus-mediated in vitro plant regeneration reducing the regeneration potential due to the aging of the calli has been overcome.
PubMed: 37853411
DOI: 10.1186/s13007-023-01081-3