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The Emerging Role of Exosome Nanoparticles in Regenerative Medicine
Published in Harishkumar Madhyastha, Durgesh Nandini Chauhan, Nanopharmaceuticals in Regenerative Medicine, 2022
Zahra Sadat Hashemi, Mahlegha Ghavami, Saeed Khalili, Seyed Morteza Naghib
Methods for accommodation of small molecules, protein, or nucleic acids into the exosomes are: (1) physical methods such as electroporation (in the range of 150–700 V), sonication, extrusion procedures, freeze–thaw cycles, incubation at RT (with or without the use of saponin permeabilisation), or other temperature. The small-sized molecules can cross the lipid bilayer of exosome by simple incubation. (2) Chemical methods such as transfection by Lipofectamine 2000. This method is frequently used for siRNA (small interference RNA) packaging into the exosomes. (3) Biological methods called transfection of exosome-producing cells. In this method, the parent cells are genetically modified to overexpress a certain gene. The overexpressed gene would ultimately be collected into the parent cell-derived exosomes. For example, various sources of Mesenchymal Stem Cells (MSCs) were transfected with miR-146b and the exosomes containing the miR-146a cargo were collected from culture media (Johnsen et al. 2014).
Application of Bioresponsive Polymers in Gene Delivery
Published in Deepa H. Patel, Bioresponsive Polymers, 2020
Tamgue Serges William, Drashti Pathak, Deepa H. Patel
Mao et al. (2007) have successfully synthesized a thermoresponsive copolymer made up of trimethyl CS-grafted by poly(N-isopropyl acrylamide) (TMC-g-PNIPAAm) with an LCST (32°C). They showed a strong ability to combine with DNA at 40°C and optimized gene transfection efficiency comparable to Lipofectamine 2000, while no obvious cytotoxicity has been observed. Some other example of thermosensitive polymers which can be exploited as carriers for therapeutics nucleic acid delivery includes:
Gold Nanomaterials at Work in Biomedicine *
Published in Valerio Voliani, Nanomaterials and Neoplasms, 2021
Xuan Yang, Miaoxin Yang, Pang Bo, Madeline Vara, Younan Xia
Electrostatic interaction is another effective means for loading a drug onto the surface of Au nanoparticles. In one demonstration, CTAB-stabilized Au nanorods were utilized as a platform for the delivery of small interfering RNA (siRNA) and single-stranded RNA (ssRNA) [589, 590]. Because of the strong electrostatic interaction between CTAB-stabilized Au nanorods (positively charged) and nucleic acid (negatively charged), the nucleic acid molecules could be readily immobilized on the surface of Au nanorods. The resultant system caused gene silencing with no observed cytotoxicity to the cells as shown by the reduction in expression of some key proteins. In another approach, layer-by-layer (LbL) assembly based on electrostatic interactions between positively and negatively charged species was utilized to add multiple layers of polymers onto the surface of a nanoparticle [582, 591–594]. The LbL method is simple and versatile, and various types of charged molecules can be incorporated. In a typical process, charge-reversed polyelectrolytes such as PEI and poly(allylamine hydrochloride)–citraconic anhydride (PAH-Cit) were used to functionalize Au nanoparticles for the delivery of drugs such as siRNA [582]. The use of functionalized Au nanoparticles improved the expression efficiency of enhanced green fluorescent protein (EGFP), and showed much lower toxicity toward cell proliferation. Compared to commercial Lipofectamine 2000, the system based on Au nanoparticles also showed better knockdown efficiency [582]. The functionalized Au nanoparticles could protect siRNA against enzymatic degradation at a vector to RNA mass ratio of 2.5:1 and above. Using this carrier, the uptake of siRNA by HeLa cells could be significantly improved relative to PEI, which is an efficient polycationic transfection reagent. Due to the simplicity and efficiency of the LbL approach, many other polyelectrolytes have also been used for the functionalization of Au nanoparticles for drug delivery [592, 593].
Ultrasound-targeted microbubble destruction mediated miR-492 inhibitor suppresses the tumorigenesis in non-small cell lung cancer
Published in Annals of Medicine, 2021
Wendi Zou, Yan Wang, Qingqing Song, Qianqian Li, Jie Ren, Xiaoyu Liu, Wei Cui
Lipofectamine, which has been widely and commonly used for gene transfection into cultured cells [38]. The study by Shi et al. showed that a high level of transfection and transduction efficiency using Lipofectamine 3000 transfection reagent compared with Lipofectamine 2000 or FuGENE 6 reagents could be achieved [39]. In addition, studies have confirmed that Lipofectamine 3000™ had the least impact on cell morphology and viability [40]. Thus, UTMD and Lipofectamine 3000 (Invitrogen, California, USA) were combined in this study. Based on the cell experimental results, we speculated that both approaches may yield a synergistic utility such that cell transfection efficiencies WERE superimposed to significantly improve transfection efficiency. However, the efficiency of the UTMD technique was affected by the ultrasound irradiation conditions. Thus, the application of UTMD needs to be explored under conditions with different loading products and different cell strains.
OB-RGRP regulates the phosphorylation of JAK2 and STAT3 in primary rat adipocytes
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Xiaolin Li, Weihong Shi, Guohui Wu, Xu Qin, Guanqun Wan, Qi Zeng, Yungang Hu
Dulbecco's Modified Eagle Medium: nutrient mixture F-12 (DMEM/F-12) media and fetal bovine serum (FBS) were bought from Gibco (Carlsbad, CA, USA). Lipofectamine 3000 reagent was purchased from Invitrogen (Carlsbad, CA, USA). Type I collagenase was obtained from Solarbio (Beijing, China). Oil red O staining kit was gotten from KeyGen Biotech (Nanjing, Jiangsu, China). EcoRI and XhoI were bought from Thermo Scientific (Waltham, MA, USA). Trizon reagent, Ultrapure RNA extraction kit, HiFiScript cDNA synthesis kit and UltraSYBR Mixture were bought from CWBIO (Beijing, China). Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (TA-08) and horseradish peroxidase (HRP) conjugated goat anti-mouse IgG(H + L) (ZB-2305), HRP-conjugated goat anti-rabbit IgG(H + L) (ZB-2301) were obtained from ZSGB-BIO (Beijing, China). Rabbit anti-OB-RGRP polyclonal antibody (DF7139) was gotten from Affinity (Cincinnati, OH, USA). Rabbit anti-signal transducer and activator of transcription 3 (STAT3) polyclonal antibody (A1192) was obtained from ABclonal (Wuhan, Hubei, China). Rabbit anti-Janus kinase 2 (JAK2) monoclonal antibody (ab108596), rabbit anti-phosphorylated JAK2 (p-JAK2) monoclonal antibody (ab32101) and rabbit anti-phosphorylated STAT3 (p-STAT3) monoclonal antibody (ab32143) were bought from Abcam (Cambridge, MA, USA).
BRCA2 protects mammalian cells from heat shock
Published in International Journal of Hyperthermia, 2018
Yosuke Nakagawa, Atsuhisa Kajihara, Akihisa Takahashi, Akiho S. Murata, Masaya Matsubayashi, Soichiro S. Ito, Ichiro Ota, Takahiko Nakagawa, Masatoshi Hasegawa, Tadaaki Kirita, Takeo Ohnishi, Eiichiro Mori
The siRNA sequence used for human BRCA2 was AAC AAC AAU UAG GAA CCA AAC UU [23]. The siRNA sequence of the non-specific negative control was the same as used previously [24]. The siRNA duplexes were synthesised and provided as a purified and annealed duplex by the Japan Bio Services Co., Ltd. (Saitama, Japan). Human BRCA2 siRNA or a non-specific negative control siRNA was transfected into human tongue squamous cell carcinoma SAS cells as previously described [22]. The siRNA sequences targeting BRCA2 used here are the most commonly used in other reported work. Transfections were performed using Lipofectamine RNAiMAX. Cells were seeded at 1–5 × 104 cells per 6 cm plate for 16–24 h without antibiotics. The siRNA was diluted in Opti-MEM I (Invitrogen, Carlsbad, CA) to produce a final siRNA concentration of 10 nM in a 1 ml final transfection volume. In a separate tube, 10 μl of Lipofectamine RNAiMAX was added to 490 μl of Opti-MEM I. The Lipofectamine RNAiMAX dilution was mixed with the diluted siRNA and incubated at room temperature for 15 min. The complex was then added drop-wise onto the cells. The cells were incubated for 48 h before further processing. The cells were then trypsinised and used for colony forming assays or western blot analysis.