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Plant-Derived Edible Nanoparticles in Cancer Drug Delivery
Published in Hala Gali-Muhtasib, Racha Chouaib, Nanoparticle Drug Delivery Systems for Cancer Treatment, 2020
Siavash Iravani, Ghazaleh Jamalipour Soufi
For the isolation of PDENs, differential ultracentrifugation plus density gradient centrifugation can be applied. Plants are usually ground to juice in a mixer; low-speed centrifugation is applied to remove large particles and plant fibers; medium-speed centrifugation is used to remove large debris and intact organelles; and high-speed centrifugation is used to pellet the exosome-like NPs. The quantity and quality of the obtained NPs are sensitive to the g force rotor type, and angle of rotor sedimentation. Moreover, this method sediments other vesicles, proteins and/or protein/RNA aggregates. Therefore, a subsequent sucrose density gradient step is used to separate the PDENs from contaminants of different densities. Gradient ultracentrifugation requires an extended centrifugation time (about 1–5 h), but provides a more purified edible plant nanoparticle isolate than ultracentrifugation alone [5–7].
Intrauterine Insemination with Homologous Sperm
Published in Botros Rizk, Ashok Agarwal, Edmund S. Sabanegh, Male Infertility in Reproductive Medicine, 2019
Semen preparation techniques (SPTs) are used in ART programs to select sperm cells with intact functional and genetic properties, including normal morphology, minimal DNA damage, and intact cell membranes with functional binding properties [31]. The most commonly used techniques are the density-gradient centrifugation (DGC) and swim-up technique. The DGC technique uses centrifugation to separate fractions of spermatozoa based upon their motility, size, and density. The mature, leukocyte-free spermatozoa are separated from the immature immotile sperm and are then centrifuged. However, the process of centrifugation itself can provoke leukocytes to generate high levels of reactive oxygen species (ROS). By reducing the centrifugation time rather than the centrifugation force the generation of ROS can be minimized, which may help in the retrieval of the highest proportion of mature sperm. In the swim-up technique highly motile sperm are separated based on their natural ability to migrate against gravity. This technique may be inappropriate for semen samples that contain a high concentration of ROS producer cells such as leukocytes and immature and damaged spermatozoa [31]. According to a Cochrane Review no significant differences in pregnancy rates between these two techniques in the setting of AIH could be found [32].
The LH/hCG Receptor
Published in Mario Ascoli, Luteinizing Hormone Action and Receptors, 2019
Patrick C. Roche, Robert J. Ryan
Purification of the LH/hCG receptor has been hampered by its low concentration in tissues and its instability. Little can be done about the former, but attempts have been made to increase receptor stability during purification procedures. Many investigators have experienced substantial losses of receptor activity during initial solubilization.18,36,236,238 Inclusion of protease inhibitors during extraction has improved recovery and stability.174,236,244 Addition of the structure-stabilizing agent glycerol greatly enhances the stability of soluble receptors.97,242 Following solubilization, most purification schemes have employed traditional biochemical techniques. Density gradient centrifugation, gel filtration, ion exchange, and affinity chromatography have been employed with varying degrees of success.
Promising RNA-based cancer gene therapy using extracellular vesicles for drug delivery
Published in Expert Opinion on Biological Therapy, 2020
Vivian Weiwen Xue, Sze Chuen Cesar Wong, Guoqi Song, William Chi Shing Cho
EVs show broad prospects in cancer gene therapy. However, there are still many unknowns about the function and working mechanism of EVs. Firstly, there is no standardized method for isolation, purification, and quantification of EVs. For isolation of EVs, differential centrifugation, density-gradient centrifugation, and immunoaffinity are the three most common methods. However, differential centrifugation and density-gradient centrifugation are time-consuming and do not ensure the purity of isolated EVs. Immunoaffinity provides better isolation and purification to separate subpopulations of EVs, but the process is complex and costly [57]. In addition, EVs from different cell sources or donors have significant differences in size, shape, and cargos. This increases the difficulty of quality control for EVs production. For example, mesenchymal stem cells (MSCs) and DCs are the most common sources of EVs and drug endogenous loading [11,56]. However, until now there is no effective way to control or limit the natural cargos and the efficiency of pharmaceutical packaging for EVs generated from MSCs and DCs. Besides, the preservation of EVs and therapeutic RNA is another challenge, and the temperature lower than −80°C is commonly needed to avoid degradation [51].
DNA released from neutrophil extracellular traps (NETs) activates pancreatic stellate cells and enhances pancreatic tumor growth
Published in OncoImmunology, 2019
Jennifer L. Miller-Ocuin, Xiaoyan Liang, Brian A. Boone, W. Reed Doerfler, Aatur D. Singhi, Daolin Tang, Rui Kang, Michael T. Lotze, Herbert J. Zeh
Under sterile conditions, bone marrow neutrophils were isolated from the femur and tibia of euthanized mice by the previously described protocol.56 Bones were cleaned with 70% ethanol and rinsed with ice-cold PBS. Bone marrow was flushed from bones using RPMI supplemented with 10% heat-inactivated fetal bovine serum and 2mM EDTA using a 27.5 gauge needle. Cells were collected into a 50 mL conical tube through a 100um filter. Following centrifugation at 1400 rpm for 7 min at 4°C red blood cells were subjected to hypotonic lysis with 0.2% NaCl for 20 s. Cells were then collected by centrifugation, washed in supplemented RPMI, centrifuged, and then resuspended in ice-cold PBS. Density gradient centrifugation was performed with Histopaque 1119 and 1077. Neutrophils were collected from the gradient interface, washed twice, and counted.
Prevention of HIV transmission with sperm washing within fertile serodiscordant couples undergoing non-stimulated intrauterine insemination
Published in AIDS Care, 2021
Waldemar de Almeida Pereira de Carvalho, Edir Catafesta, Itatiana Ferreira Rodart, Silvio Takata, Denise Lotufo Estevam, Caio Parente Barbosa
Sperm washing was conducted under sterile conditions. The whole semen sample was transferred to a sterile conical centrifuge tube, and homogenized with the same amount of HEPES-buffered human tubal fluid medium (mHTF, Irvine Scientific, Santa Ana, CA, USA) supplement with 10% synthetic serum substitute (SSS), and centrifuged for 10 min at 1600 rpm. The supernatant was then removed and the pellet suspended in up to 1.5 mL of mHTF 10% SSS. Density gradient centrifugation technique was then performed. Using a sterile pipette, 1.0 mL of the “lower layer” (90% Isolate, Irvine Scientific, Santa Ana, CA, USA) was transferred into a conical centrifuge tube. Using a new sterile pipette 1.0 mL of the “upper layer” (45% Isolate, Irvine Scientific, Santa Ana, CA, USA) was gently dispensed on top of the lower layer. The 1.5 mL washed semen sample was then placed on top of the upper layer and the tube was centrifuged for 20 min at 1600 rpm. The upper and lower layers were removed using a plastic pipette. Then, 1.0 mL of mHTF 10% SSS was added to the tube and the re-suspended pellet was centrifuged for 10 min at 1600 rpm. The supernatant was removed and the pellet was re-suspended in 1.0 mL of mHTF 10% SSS. Finally, the re-suspended pellet was gently added to the bottom of a conical tube containing 1 mL mHTF 10% SSS. The closed tube, inclined at 45° was kept at room temperature for 40 min, when a sterile Pasteur pipette was used to aspirate 0.5 mL of the supernatant and transfer it to a sterile conic tube. Sperm count and motility were estimated in the recovered fraction. The swim-up procedure was not performed if sperm concentration was <5 × 106/mL.