A nanocarrier system for the delivery of nucleic acids targeted to a pancreatic beta cell line
Introduction
Diabetes mellitus is a multi-factorial disorder characterized by high blood glucose levels, and appears to be caused, in part, by genetic and environmental factors [1], [2]. Mutations of the gene related to pancreatic β cell function have been associated with some types of diabetes mellitus [3], [4]. For example, maturity-onset diabetes of the young (MODY) are caused by mutations in any one of at least six different genes that encode the glycolytic enzyme glucokinase and five transcription factors [5]. These genes are associated with important pancreatic β cell functions, including the production and secretion of insulin which regulates blood glucose levels. Therefore, gene therapy targeted to pancreatic β cells and related studies could be useful for the treatment of many patients suffering from diabetes mellitus.
A line of pancreatic β cells “MIN6” has been established from mouse insulinomas, and has morphological characteristics that are similar to those of pancreatic β cells. In addition, MIN6 cells exhibit glucose-inducible insulin secretion comparable to that of cultured normal mouse islet cells [6]. Thus, the MIN6 cell line is useful in studies of the molecular mechanisms of pancreatic β cells, and has been used in research directed to the study of the pancreatic β cells function and diabetes therapy [7], [8], [9]. The regulation of gene expression in MIN6 cells could accelerate these studies, but an efficient method for the transfection of nucleic acids targeted to MIN6 cells is required. To date, while the transfection of nucleic acids into MIN6 cells using a viral vector [9] and electroporation [8] has been achieved, the transfection efficiency does not appear to be sufficient. Considering such a situation, a nanocarrier system capable of delivering nucleic acids targeted to MIN6 cells is essential for future research on the function of β cells and diabetes therapy.
In previous studies, we reported on the development of a multifunctional envelope-type nano device (MEND), which consists of a condensed plasmid DNA (pDNA) core and lipid envelopes [10], [11], which showed transfection activities as high as that for a viral vector in dividing cells [12]. To date, we have been successful in efficiently packaging nucleic acids, including oligo DNA [13], [14] and siRNA [15], [16], and showed that the MEND system achieved efficient gene silencing and RNA knockdown [14], [15], [16], [17], [18], [19]. These results prompted us to consider the possibility that a MEND system could be used to achieve the efficient cytosolic delivery of nucleic acids targeted to impregnable MIN6 cells.
In this study, we report on our attempts to develop β-MEND, which is a MEND that permits the efficient delivery of nucleic acids to pancreatic β cells. We conclude that the β-MEND reported on here, constitutes a breakthrough in research on the function of β cells and diabetes therapy. We first identified a lipid composition for the β-MEND that permits it to be efficiently internalized into MIN6 cells. This was achieved by varying the lipid composition of a panel of liposomes (LPs) labeled with fluorescent lipids and cellular uptake analysis by flow cytometry. We next constructed the β-MEND in which nucleic acids were encapsulated, and the fluorescent labeled nucleic acids that were internalized in MIN6 by the β-MEND were measured by flow cytometry. We also observed the intracellular trafficking of the nucleic acids using confocal laser scanning microscopy (CLSM). Moreover, when a β-MEND encapsulating a 2′-O-Methyl (2′-OMe) RNA which targets a microRNA (miRNA) that suppresses insulin secretion was used, the knockdown of the targeted miRNA and the up-regulation of insulin secretion were observed and evaluated in MIN6 cells.
Section snippets
Materials
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilawroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg yolk phosphatidyl choline (EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) were obtained from Nippon Oil and Fats Co. (Tokyo, Japan). Cardiolipin (CL), cholesteryl hemisuccinate (CHEMS), phosphatidic acid (PA) and
Screening for a lipid composition with high affinity to MIN6 cells
We recently developed a MEND, consisting of a condensed pDNA core and lipid envelopes [10], [11], the surface of which was modified with high-density R8, an artificially designed cell penetrating peptide [20], [22], to produce the R8-MNED. A previous report showed that high-density R8-modified carriers were internalized primarily via macropinocytosis rather than clathrin-mediated endocytosis, as is the case of cationic LPs [23], [24]. Moreover, the R8-MEND showed transfection activities in
Discussion
In this study, we report on attempts to develop a nucleic acid carrier consisting of a unique lipid envelope (referred to herein as a “β-MEND”) targeted to MIN6 cells. The MIN6 cell line has been used in research on the function of β cells and diabetes therapy [7], [8], [9], thus a nucleic acid delivery system targeted to MIN6 cells could accelerate our understanding in these fields. We focused on the development of a non-viral vector, R8-MEND [10], [11] with a high transfection activity
Conclusion
In this study, we successfully identified a lipid composition (DC-Chol/EPC/SM = 3:4:3, molar ratio) for the β-MEND that comprised a high affinity envelope for MIN6 cells, model pancreatic β cells. Flow cytometry analysis and intracellular observations by CLSM indicated that the β-MEND efficiently delivered nucleic acids to the cytosol in MIN6 cells. Moreover, the transfection of 2′-OMe RNA complementary to miR-375 into MIN6 cells by the β-MEND system resulted in the efficient knockdown of the
Acknowledgments
This work was supported, in part by, the Program for Promotion of Fundamental Studies in Health Sciences (project ID 10-62) of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Scientific Research (S) (grant number 21229002) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We thank Dr. J. Miyazaki for providing MIN6 cells. We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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