While this can help attribute particle uptake to specific pathways like caveolae-mediated or clathrin-mediated endocytosis, this does not provide a holistic picture of the delivery process. membrane permeability. By applying this strategy to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guideline RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery. transcribed single guideline RNA, sgRNA (synthetic can also be used) pSPCas9 (Addgene, px165) Opti-MEM reduced serum medium (Thermo Fisher, Gibco, catalog quantity: 31985062) Dulbeccos altered Eagle medium, DMEM (Thermo Fisher, Gibco, catalog quantity: 11960077) Fetal bovine serum (Thermo Fisher, Gibco, catalog quantity: 16000044) Penicillin-streptomycin (Thermo Fisher, Gibco, catalog quantity: 15140148) Phosphate buffered saline, or PBS (Fisher Scientific, Corning, catalog quantity: 21040CV) Delivery vehicle such as a helical nanoparticle (HNP) formulated from poly(transcribed sgRNA by using the HiScribe T7 Large Yield RNA Synthesis Kit and subbing Fluorescein-12-UTP for the un-tagged UTP at a specific percentage. To determine the percentage of Fluorescein-12-UTP to UTP, determine the number of Uracils in the sgRNA sequence and, based off of 2 fluorescein per sgRNA strand, add Fluorescein-12-UTP to UTP in the percentage of 2 to (total number of UTP) -2. is the slope TC-S 7010 (Aurora A Inhibitor I) derived from fitting the data to a linear curve of concentration versus fluorescence and is the Y-intercept. X = Y + em a /em X = 61800Y + 1100 Y = 1/(61800) * (X-1100) Then we can calculate the percent uptake and inhibition as with Number 3, with the following fluorescence readings from Methods A, C, D, as with the 1st column below: Open in a separate window Number 3. Cell membrane permeability.Use the amount of FITC-Tris in lysate to correlate to membrane permeability. The more FITC-Tris able to cross into the cell, the more permeability the nanoparticle (or any cell treatment) induced in the cell membrane. Here UT refers to the untreated group, while Free FITC-Tris is an untreated group exposed to the FITC-Tris; HNP and P-HNP are helical nanoparticles and PEGylated helical nanoparticles respectively. thead th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Sample /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Fluorescence (rfu) /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ FITC TC-S 7010 (Aurora A Inhibitor I) conc. (g/ml) /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Normalized Uptake (%) /th th style=”border-bottom: 0.5px solid;” rowspan=”1″ colspan=”1″ Inhibition (%) /th /thead A23000.0194171000B18000.01132758.3333341.66667C15000.00647233.3333366.66667 Open in a separate window Recipes Cell culture medium 500 ml of Dulbeccos modified Eagles medium 5 ml of Penicillin-streptomycin (10,000 U/ml) 50 ml of fetal bovine serum HNPs composed of PPABLG (delivery vehicles) em Notice: PPABLG is just a polymer that can be used to formulate helical nanoparticles with genetic cargo and is only an example of a delivery platform that you can use to observe the intracellular trafficking and uptake in cells. Some simpler vehicles that can be directly purchased are materials like Silica Oxide nanoparticles or Lipofectamine. /em Inside a glovebox, 1st add -(4-vinylbenzyl)-L-glutamate N-carboxyanhydride (VB-L-Glu-NCA) and dissolve in anhydrous dimethyl formamide (DMF), then add a DMF answer of hexamethyldisilazane (M/I = 200) and nitrobenzene The polymerization is definitely carried out at room heat until the conversion of NCA reached 99% (monitored by Fourier Transform Infrared Spectroscopy). Purify the polypeptide precursor, poly( em /em -(4-vinyl)benzyl-L-glutamate) (PVBLG), by precipitation in hexane:ether (1:1, v/v) Then dissolve PVBLG in chloroform, TC-S 7010 (Aurora A Inhibitor I) and the side-chain vinyl organizations are oxidized into aldehydo organizations by bubbling O3 gas into the answer at -78 C Purify the producing polypeptide, poly( em /em -(4-aldehydo)benzyl-L-glutamate) (PABLG) by precipitation in methanol, and analyze by 1 H NMR to confirm the conversion of side-chain vinyl groups Then Dissolve PABLG in DMF, into which 1-(2-aminoethyl) piperidine and borane-pyridine complex are added sequentially to react with the side-chain aldehyde groups of PABLG Purify the final product PPABLG by dialysis against DI water and then lyophilize to yield white powder 4% paraformaldehyde 25 ml of paraformaldehyde 75 ml of PBS Acknowledgments The research was supported from the NIH (Grants nos. HL109442, AI096305, GM110494, and UH3 TR000505). Competing interests The authors declare no conflicts of interest or competing interests. Citation Readers should cite both the Bio-protocol article and the original research article Mouse monoclonal to CD4/CD8 (FITC/PE) where this protocol was used..The more FITC-Tris able to cross into the cell, the more permeability the nanoparticle (or any cell treatment) induced in the cell membrane. delivery process. Here, we provide a general protocol that combines systematic studies of inhibitor effects on effectiveness with quantification of particle-induced cell membrane permeability. By applying this strategy to a nucleic acid delivery system, for example a helical polypeptide-based nanoparticle for plasmid and guideline RNA delivery, we gain understanding of the endocytic mechanisms and cell uptake for intelligent design of intracellular delivery. transcribed solitary lead RNA, sgRNA (synthetic can also be used) pSPCas9 (Addgene, px165) Opti-MEM reduced serum medium (Thermo Fisher, Gibco, catalog quantity: 31985062) Dulbeccos altered Eagle medium, DMEM (Thermo Fisher, Gibco, catalog quantity: 11960077) Fetal bovine serum (Thermo Fisher, Gibco, catalog quantity: 16000044) Penicillin-streptomycin (Thermo Fisher, Gibco, catalog quantity: 15140148) Phosphate buffered saline, or PBS (Fisher Scientific, Corning, catalog quantity: 21040CV) Delivery vehicle such as a helical nanoparticle (HNP) formulated from poly(transcribed sgRNA by using the HiScribe T7 Large Yield RNA Synthesis Kit and subbing Fluorescein-12-UTP for the un-tagged UTP at a specific percentage. To determine the percentage of Fluorescein-12-UTP to UTP, determine the number of Uracils in the sgRNA sequence and, based off of 2 fluorescein per sgRNA strand, add Fluorescein-12-UTP to UTP in the percentage of 2 to (total number of UTP) -2. is the slope derived from fitting the data to a linear curve of concentration versus fluorescence and is the Y-intercept. X = Y + em a /em X = 61800Y + 1100 Y = 1/(61800) * (X-1100) Then we can calculate the percent uptake and inhibition as with Number 3, with the following fluorescence readings from Methods A, C, D, as with the 1st column below: Open in a separate window Number 3. Cell membrane permeability.Use the amount of FITC-Tris in lysate to correlate to membrane permeability. The more FITC-Tris able to cross into the cell, the more permeability the nanoparticle (or any cell treatment) induced in the cell membrane. Here UT refers to the untreated group, while Free FITC-Tris is an untreated group exposed to the FITC-Tris; HNP and P-HNP are helical nanoparticles and PEGylated helical nanoparticles respectively. thead th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Sample /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Fluorescence (rfu) /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ FITC conc. (g/ml) /th th style=”border-bottom: 0.5px solid; border-right: 0.5px solid;” rowspan=”1″ colspan=”1″ Normalized Uptake (%) /th th style=”border-bottom: 0.5px solid;” rowspan=”1″ colspan=”1″ Inhibition (%) /th /thead A23000.0194171000B18000.01132758.3333341.66667C15000.00647233.3333366.66667 Open in a separate window Recipes Cell culture medium 500 ml of Dulbeccos modified Eagles medium 5 ml of Penicillin-streptomycin (10,000 U/ml) 50 ml of fetal bovine serum HNPs composed of PPABLG (delivery vehicles) em Notice: PPABLG is just a polymer that can be used to formulate helical nanoparticles with genetic cargo and is only an example of a delivery platform that you can use to observe the intracellular trafficking and uptake in cells. Some simpler vehicles that can be directly purchased are materials like Silica Oxide nanoparticles or Lipofectamine. /em Inside a glovebox, 1st add -(4-vinylbenzyl)-L-glutamate N-carboxyanhydride (VB-L-Glu-NCA) and dissolve in anhydrous dimethyl formamide (DMF), then add a DMF answer of hexamethyldisilazane (M/I = 200) and nitrobenzene The polymerization is definitely carried out at room heat until the conversion of NCA reached 99% (monitored by Fourier Transform Infrared Spectroscopy). Purify the polypeptide precursor, poly( em /em -(4-vinyl)benzyl-L-glutamate) (PVBLG), by precipitation in hexane:ether (1:1, v/v) Then dissolve PVBLG in chloroform, and the side-chain vinyl organizations are oxidized into aldehydo organizations by bubbling O3 gas into the answer at -78 C Purify the producing polypeptide, poly( em /em -(4-aldehydo)benzyl-L-glutamate) (PABLG) by precipitation in methanol, and analyze by 1 H NMR to confirm the conversion of side-chain vinyl groups Then Dissolve PABLG in DMF, into which 1-(2-aminoethyl) piperidine and borane-pyridine complex are added sequentially to react with the side-chain aldehyde groups of PABLG Purify the final product PPABLG by dialysis against DI water and then lyophilize to yield white powder 4% paraformaldehyde.