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Optimized Conditions for the Delivery of Small Membrane Impermeable Compounds into Human Cells Using Hypotonic Shift

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DOI: 10.4236/cellbio.2012.12005    4,105 Downloads   8,304 Views  

ABSTRACT

Cell-based assays represent a major end point of high throughput screening (HTS) but a key limitation of such assays is the potentially poor membrane permeability of test compounds. In this study, we optimized the conditions for the delivery of the membrane impermeable compound 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) into human cells using hypotonic shift; a method that can promote the uptake of molecules from the extracellular fluid into cell cytoplasm via endocytosis. We showed that uptake of HPTS by cells was a function of hypotonic buffer osmolarity and that delivery was highly efficient with almost 100% of cells displaying uptake. Delivery of HPTS was equally effective at 25°C and 37°C, with delivery of compound proportional to incubation time and concentration of HPTS within the loading medium. The experimental conditions identified in this study could be applied to HTS drug discovery studies providing an effective method of delivering small membrane impermeable compounds into cells.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

A. Stephens, C. Day and J. Tiralongo, "Optimized Conditions for the Delivery of Small Membrane Impermeable Compounds into Human Cells Using Hypotonic Shift," CellBio, Vol. 1 No. 2, 2012, pp. 38-42. doi: 10.4236/cellbio.2012.12005.

References

[1] E. A. Martis, R. Radhakrishnan and R. R. Badve, “High-Throughput Screening: The Hits and Leads of Drug Discovery An Overview,” Journal of Applied Pharmaceutical Science, Vol. 1, No. 1, 2011, pp. 2-10.
[2] W. F. An and N. Tolliday, “Cell-Based Assays for High-Throughput Screening,” Molecular Biotechnology, Vol. 45, No. 2, 2010, pp. 180-186. doi:10.1007/s12033-010-9251-z
[3] B. S. Gan, E. Krump, L. D. Shrode and S. Grinstein, “Loading Pyranine via Purinergic Receptors or Hypotonic Stress for Measurement of Cytosolic pH by Imaging,” American Journal of Physiology, Vol. 275, No. 4, 1998, pp. C1158-C1166.
[4] K. Koberna, D. Stanek, J. Malinsky, M. Eltsov, A. Pliss, V. Ctrnacta, S. Cermanova and I. Raska, “Nuclear Organization Studied with the Help of a Hypotonic Shift: Its Use Permits Hydrophilic Molecules to Enter into Living Cells,” Chromosoma, Vol. 108, No. 5, 1999, pp. 325-335. doi:10.1007/s004120050384
[5] I. Kuriyama, T. Mizuno, K. Fukudome, K. Kuramochi, K. Tsubaki, T. Usui, N. Imamoto, K. Sakaguchi, et al., “Effect of Dehydroaltenusin-C12 Derivative, a Selective DNA Polymerase Alpha Inhibitor, on DNA Replication in Cultured Cells,” Molecules, Vol. 13, No. 12, 2008, pp. 2948-2961. doi:10.3390/molecules13122948
[6] Y. H. Lee and C. A. Peng, “Effect of Hypotonic Stress on Retroviral Transduction,” Biochemical and Biophysical Research Communications, Vol. 390, No. 4, 2009, pp. 1367-1371. doi:10.1016/j.bbrc.2009.10.161
[7] J. L. Lemoine, R. Farley and L. Huang, “Mechanism of Efficient Transfection of the Nasal Airway Epithelium by Hypotonic Shock,” Gene Therapy, Vol. 12, No. 16, 2005, pp. 1275-1282. doi:10.1038/sj.gt.3302548
[8] J. Sharif, M. Muto, S. Takebayashi, I. Suetake, A. Iwamatsu, T. A. Endo, J. Shinga, Y. Mizutani-Koseki, et al., “The SRA Protein Np95 Mediates Epigenetic Inheritance by Recruiting Dnmt1 to Methylated DNA,” Nature, Vol. 450, No. 7171, 2007, pp. 908-912. doi:10.1038/nature06397
[9] D. Stanek, K. Koberna, A. Pliss, J. Malinsky, M. Masata, J. Vecerova, M. C. Risueno and I. Raska, “Non-Isotopic Mapping of Ribosomal RNA Synthesis and Processing in the Nucleolus,” Chromosoma, Vol. 110, No. 7, 2001, pp. 460-470. doi:10.1007/s00412-001-0172-2
[10] S. Takebayashi, T. Tamura, C. Matsuoka and M. Okano, “Major and Essential Role for the DNA Methylation Mark in Mouse Embryogenesis and Stable Association of DNMT1 with Newly Replicated Regions,” Molecular and Cellular Biology, Vol. 27, No. 23, 2007, pp. 8243-8258. doi:10.1128/MCB.00899-07
[11] E. K. Hoffmann, I. H. Lambert and S. F. Pedersen, “Physiology of Cell Volume Regulation in Vertebrates,” Physiological Reviews, Vol. 89, No. 1, 2009, pp. 193-277. doi:10.1152/physrev.00037.2007
[12] W. C. O’Neill, “Physiological Significance of Volume-Regulatory Transporters,” American Journal of Physiology, Vol. 276, No. 5, 1999, pp. C995-C1011.
[13] T. van der Wijk, S. F. Tomassen, A. B. Houtsmuller, H. R. de Jonge and B. C. Tilly, “Increased Vesicle Recycling in Response to Osmotic Cell Swelling. Cause and Consequence of Hypotonicity-Provoked ATP Release,” The Journal of Biological Chemistry, Vol. 278, No. 41, 2003, pp. 40020-40025. doi:10.1074/jbc.M307603200

  
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