The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.
Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.
Neumann
Sebastian
S
Department of Biochemistry, Molecular Neurobiochemistry, University of Bochum, Universitaetsstrasse 150, D-44780 Bochum, Germany.
Kovtun
Anna
A
Dietzel
Irmgard D
ID
Epple
Matthias
M
Heumann
Rolf
R
D002118:Calcium / Q000378:metabolism*; D002130:Calcium Phosphates / Q000737:chemistry*; D002132:Calcium Radioisotopes; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002470:Cell Survival; D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000235:genetics*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D018715:Microscopy, Video; D053758:Nanoparticles / Q000032:analysis* / Q000737:chemistry; D010316:Particle Size; D013997:Time Factors; D014162:Transfection / Q000379:methods*; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology
D002130:Calcium Phosphates; D002132:Calcium Radioisotopes; D005456:Fluorescent Dyes; D004247:DNA; C020243:calcium phosphate; D002118:Calcium; D016257:Fura-2