Tack F, Noppe M, Van Dijck A, Dekeyzer N, Van Der Leede B J, Bakker A, Wouters W, Janicot M, Brewster M E
Department of Molecular Cell Biology, Research Institute of Growth and Development, University of Maastricht, Maastricht, The Netherlands.
Pharmazie. 2008 Mar;63(3):221-5.
The objective of the current study was to develop cellular delivery approaches for catalytic DNA enzymes (DNAzymes) which cleave targeted messenger RNA, using vectors based on colloidal gold. The model DNAzyme was a 32mer oligonucleotide designed to specifically interact with and cleave c-myc mRNA. Colloidal gold particles were prepared by reduction of tetrachlororauric [III] acid with sodium citrate. Particles could be produced in the 1-90 nm range. A cationic substrate linked to transferrin was electrostatically/hydrophobically bound to the gold particle. These vectors were then treated with the DNAzyme to yield the condensed DNA-cationic polymer-particulate product. The pH (4-11.5), the quantity of the DNAzymes (0.079-0.567 microg/probe), the cationic polymer (polylysine (PL) or polyethylenimine (PEI)) as well as the surfactant (PVP) concentration (0-0.5%) were varied to give stable constructs which decomplexed under the desired conditions (i.e., in lysosomes and at lower pH values). Cellular uptake of the FITC-labelled c-myc DNAzyme incorporated in this vector was measured using FACS analysis in human HT29 colon carcinoma cells. Data suggested that PEI gave better delivery efficiencies than PL. The use of PVP to stabilize the formed dispersions was detrimental to DNAzyme delivery when PL was used but had little effect in the PEI systems. In the best cases, delivery to 77% of the cells was possible using PEI with the PVP stabilizer and completing the DNA condensation at pH 5.5 with 0.118 microg of DNAzyme/probe. In contrast, the best conditions for PL gave only transfection to 43% of the cells (no PVP, condensed at pH 5.7 and with a loading of 0.079 microg DNAzyme/probe). The PL probe tended to be more toxic than the PEI-based systems (65% cell death in PL transfected cells compared to 22% for PEI). These results suggest that cellular targeting using colloidal gold appears feasible for DNAzyme delivery.
本研究的目的是开发基于胶体金的载体，用于催化性DNA酶（脱氧核酶）的细胞递送，该酶可切割靶向信使RNA。模型脱氧核酶是一种32聚体寡核苷酸，设计用于与c-myc mRNA特异性相互作用并进行切割。通过用柠檬酸钠还原四氯金酸[III]制备胶体金颗粒。颗粒可在1-90nm范围内产生。与转铁蛋白相连的阳离子底物通过静电/疏水作用结合到金颗粒上。然后用脱氧核酶处理这些载体，得到浓缩的DNA-阳离子聚合物-颗粒产物。改变pH值（4-11.5）、脱氧核酶的量（0.079-0.567μg/探针）、阳离子聚合物（聚赖氨酸（PL）或聚乙烯亚胺（PEI））以及表面活性剂（聚乙烯吡咯烷酮（PVP））浓度（0-0.5%），以得到在所需条件下（即在溶酶体中及较低pH值下）解聚的稳定构建体。在人HT29结肠癌细胞中，使用荧光激活细胞分选（FACS）分析测量了掺入该载体中的异硫氰酸荧光素（FITC）标记的c-myc脱氧核酶的细胞摄取情况。数据表明，PEI比PL具有更好的递送效率。当使用PL时，使用PVP稳定形成的分散体对脱氧核酶的递送不利，但在PEI系统中影响较小。在最佳情况下，使用带有PVP稳定剂的PEI，并在pH 5.5下用0.118μg脱氧核酶/探针完成DNA浓缩，可将77%的细胞进行递送。相比之下，PL的最佳条件下仅能转染43%的细胞（无PVP，在pH 5.7下浓缩，加载量为0.079μg脱氧核酶/探针）。PL探针往往比基于PEI的系统毒性更大（PL转染细胞中有65%的细胞死亡，而PEI为22%）。这些结果表明，使用胶体金进行细胞靶向递送脱氧核酶似乎是可行的。
