Brus Carola, Kleemann Elke, Aigner Achim, Czubayko Frank, Kissel Thomas
Department of Pharmaceutics and Biopharmacy, Philipps-University Marburg, Ketzerbach 63, 35037 Marburg, Germany.
J Control Release. 2004 Feb 20;95(1):119-31. doi: 10.1016/j.jconrel.2003.10.021.
In the present study the lyophilization of oligodeoxynucleotide-polyethylenimine (ODN-PEI) complexes was investigated regarding the maintenance of physicochemical properties and influence on biological activity. To achieve this, we used PEI of different molecular weights, in the range of 800-0.8 kDa, as complexing agents for unmodified ODN and ribozymes. The hydrodynamic diameter was measured by photon correlation spectroscopy (PCS) and the zeta potential was determined using laser Doppler anemometry (LDA) of ODN complexes with PEI derivatives of different molecular weights both before and after lyophilization. Atomic force microscopy (AFM) was used to visualize freshly prepared, stored and lyophilized complexes in solution. The biological activity of the ODN, as well as of plasmid DNA, in lyophilized PEI complexes was examined and compared to freshly prepared complexes using standard transfection assays. All PEI derivatives formed very small complexes with ODN displaying hydrodynamic diameters ranging from 15 to 30 nm. Marginal changes in size after lyophilization were observed for ODN-PEI complexes. In contrast, plasmid complexed with PEI was found to aggregate. In either cases minimal or no influence of the added amount of lyoprotectant was observed. The shape of the very small and highly condensed ODN complexes was not altered by lyophilization as seen in the AFM images. The transfection efficiency of lyophilized ribozyme-PEI complexes relative to freshly prepared complexes was approximately 100%, whereas a decrease was seen for lyophilized plasmid-PEI complexes. An additive of the lyoprotectants trehalose, mannitol or sucrose preserved biological activity. This study demonstrates the particular suitability of ODN-PEI complexes to be formulated as lyophilized systems with no loss in physical stability or biological activity.
在本研究中，我们对寡脱氧核苷酸 - 聚乙烯亚胺（ODN - PEI）复合物的冻干过程进行了研究，考察其物理化学性质的维持情况以及对生物活性的影响。为此，我们使用了分子量在800 - 0.8 kDa范围内的不同分子量的PEI，作为未修饰的ODN和核酶的络合剂。通过光子相关光谱法（PCS）测量流体动力学直径，并使用激光多普勒测速仪（LDA）测定冻干前后不同分子量PEI衍生物与ODN复合物的zeta电位。利用原子力显微镜（AFM）观察溶液中新鲜制备、储存和冻干后的复合物。使用标准转染试验检测并比较了冻干的PEI复合物中ODN以及质粒DNA的生物活性与新鲜制备的复合物。所有PEI衍生物与ODN形成的复合物都非常小，流体动力学直径在15至30 nm之间。观察到ODN - PEI复合物冻干后尺寸变化很小。相比之下，发现与PEI复合的质粒会聚集。在这两种情况下，均未观察到冻干保护剂添加量有最小或没有影响。如AFM图像所示，非常小且高度浓缩的ODN复合物的形状未因冻干而改变。冻干的核酶 - PEI复合物相对于新鲜制备的复合物的转染效率约为100%，而冻干的质粒 - PEI复合物的转染效率则有所下降。冻干保护剂海藻糖、甘露醇或蔗糖的添加剂可保留生物活性。本研究表明ODN - PEI复合物特别适合配制成冻干系统，且物理稳定性和生物活性均无损失。
