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Anti-HSV and Cytotoxicity Properties of Three Different Nanoparticles Derived from Indian Medicinal Plants
Published in P. Mereena Luke, K. R. Dhanya, Didier Rouxel, Nandakumar Kalarikkal, Sabu Thomas, Advanced Studies in Experimental and Clinical Medicine, 2021
K. Vasanthi, G. Reena, G. Sathyanarayanan, Elanchezhiyan Manickan
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used for the screening of extracts toxicity using standard protocol. Briefly, verocells were incubated at 37°C on 96-well plates at a density of 10,000 cells/well, with 5% CO2 in a humidified atmosphere with 10% Dulbecco’s Modified Essentials Medium. After 24 hours, nanoparticles were added on monolayer (70–80% confluency) to final concentrations of 50 µg to 10 mg. The plate was incubated for further 5 day under the same conditions mentioned above. 200 µl MTT (Sigma-Aldrich, Catalogue No. M2003) solutions (5 mg/ml in phosphate buffer) was added to each well and incubated at 37°C for 4 hours. The MTT solution was decanted off, and Formosan was extracted from the cells with 250 µl of DMSO in each well. Color was measured with a 12-well ELISA plate reader at 550 nm (Figure 13.4). Toxicity Control used as 1% Triton X-100 (Qualigens, catalog No. 10655). All MTT assays were repeated three times. Cell viability was calculated using the following formula:
Transcriptional Regulation of the Human C3 Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Gretchen J. Darlington, Deborah R. Wilson, Todd S.-C. Juan
Hep 3B2 cells were maintained as monolayer cultures in 3 MEM: 1 MAB (MEM, Eagles’s minimal essential medium; MAB, MAB 87/3; GIBCO, Gaithersburg, MD), 2% fetal bovine serum (FBS: Hazleton, Lenexa, KS), and 8% horse serum (Hazleton) and passaged weekly by trypsinization. Cells used for Northern analysis were processed as previously described.21 In experiments where cycloheximide (Sigma Chemical Co., St. Louis, MO) was employed, 5 μg/ml of the inhibitor was added to the culture medium 15 min before the addition of cytokines. Cells used for preparation of nuclear extracts were treated as reported.18 Cell extracts for luciferase assays were prepared either by the freeze-thaw procedure of DeWet et al.24 with subsequent processing as previously described18 or by Triton X-100 lysis,25 in which the cells were harvested into 300 μl of glycyl-glycine, pH 7.8, 1 mM dithiothreitol, 1% Triton X-100. Five to 30 μl of cell extract were assayed for luciferase activity in 100 mM KPB (potassium phosphate buffer), pH 7.9, 15 mM MgSO4, 5 mM ATP, using a Monolight 2010 luminometer (Analytical Luminescence Laboratories, San Diego, CA). The luminescence obtained from a 30-μl buffer sample was subtracted from each measurement prior to quantitation. Luciferase activities are expressed as relative luminescence units per milligram or per microgram cellular protein.
Evaluation of PCL/Chitosan/Nanohydroxyapatite/Tetracycline Composite Scaffolds for Bone Tissue Engineering
Published in Naznin Sultana, Sanchita Bandyopadhyay-Ghosh, Chin Fhong Soon, Tissue Engineering Strategies for Organ Regeneration, 2020
Rashid Bin Mad Jin, Naznin Sultana, Chin Fhong Soon, Ahmad Fauzi Ismail
Indirect MTT assays were performed to study the cytotoxicity of extraction medium from the scaffold specimens. The cell viability of the treatment was compared to control cells, with Triton-X 100 acting as a positive control, showing greater toxicity than the control. Figure 10.6 shows positive results toward cell viability. The results showed that cell viability was above 80% compared to the control cells. From Figure 10.6, it can be seen that PCL/CS scaffolds showed the lowest cell viability compared to the rest of the scaffolds. In addition, the incorporation of nHA boosts the rate of cell viability after 72 hours of incubation. Moreover, tetracycline-incorporated scaffolds also show the positive growth of NHOst cells. This shows that the release of TCH was not fatal with regard to cell growth.
Effect of PEGylation on drug uptake, biodistribution, and tissue toxicity of efavirenz–ritonavir loaded PAMAM G4 dendrimers
Published in Pharmaceutical Development and Technology, 2023
Rohini Kharwade, Nilesh Mahajan, Sachin More, Amol Warokar, Sachin Mendhi, Akshay Dhobley, Devendra Palve
EFV and RTV were received as generous gift samples from Emcure Pharmaceuticals Ltd. (Ahmedabad, India). Cell culture medium: RPMI-1640 media was purchased by Thermo Fisher-Gibco Life Technologies (Waltham, MA, cat no.: 11875093). Triton X 100 was obtained from Sisco Research Laboratories Pvt Ltd. (Mumbai, India). Cellulose dialysis bag MWCO 12–14 kDa, (3-(4,5-dimetylthaizol-2-2-y1)-2,5-diphenyl) tetrazolium bromide (MTT reagent) (cat no.: 4060), and fetal bovine serum (FBS) culture medium (cat no.: RM10432) were obtained and 0.45 µm membrane filter paper was purchased from Himedia Labs (Mumbai, India). 5-Fluorouracil (5FU) (cat no.: F6627), DMSO (PHR 1309), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), and used without further purification unless otherwise stated.
Synergic fabrication of multifunctional liposomes nanocomposites for improved radiofrequency ablation combination for liver metastasis cancer therapy
Published in Drug Delivery, 2022
Ning Zhang, Yibin Wu, Weiqi Xu, Zhenjian Li, Lu Wang
After the liposomes were synthesized using the thin-film dispersion technique (molar ratio 80:20:5:4), they were sized using sonography and membrane extrusion (Wang et al., 2011; Unnam et al., 2019; Xiao et al., 2021). In a round bottom flask, 25 mg of total lipids (folate-DPPC/DSPC/DSPE-PEG2000@DOX) was mixed with chloroform–diethyl ether mixture (2:1) and evaporated at 60 °C using a rotary evaporator. The resulting liposomes contained 5 mg of DOX. A rotary evaporator was used to evaporate the solvent, resulting in a thin lipid coating in the flask's circular bottom. It was then dissolved in 1 mL of phosphate buffer with 1 mg of Fe3O4-C60-PEG2000 powder (100 mM, pH 7.4). At 60 °C for 20 min, a rotary evaporator spinning at 120 rpm at atmospheric pressure dried the thin lipid layer in this suspension. Utilizing an extruder (Whatman Inc., Piscataway, NJ) to modify the liposome size, the resultant fabrication was sonicated for 10 min. It was then centrifuged for 30 min at 12,000×g in a millipore to remove DOX that had not been encapsulated. Triton X-100 was added to the filtered liposomes to cause the membrane to break. The result was then diluted with an anhydrous ethanolic solution and further sonicated to verify that DOX was fully dispersed, then centrifuged to distinct Fe3O4-C60-PEG2000@DOX.
New generation of viral nanoparticles for targeted drug delivery in cancer therapy
Published in Journal of Drug Targeting, 2022
Nikta Alvandi, Maryam Rajabnejad, Zeynab Taghvaei, Neda Esfandiari
After VLPs assembly process, VLPs should be released from the host cells by different mechanisms which depend on VLPs structure. As shown by Figure 3(B), generally for obtaining VLPs from the culture medium or i.e. purification, three steps were recorded as follows: cell lysis and clarification, intermediate purification, and polishing. The first step initiates using 1% triton X-100 after harvesting of cell culture. Triton X-100 is a detergent utilised for cell lysis and protein extraction. Then, removing cell debris and aggregates is designed as a clarification process by centrifugation. Afterward, the intermediate purification step begins that concentration has a critical role in this process. Accordingly, adsorptive chromatography is a great choice for this step. Moreover, other chromatographic matrices based on porous membrane layers are utilised too. It should be noted that these porous matrices are devoted to large particles like VLPs. In the last step, polishing, residual host-cell protein, and DNA are removed. In this step, affinity and ion-exchange chromatography are suited. Sometimes size-exclusion chromatography is used too like when VLPs-derived impurities, such as non-assembled proteins have similar electrostatic features with VLPs. After obtaining purified VLPs, ultracentrifugation is occurred in some studies [36].