Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: Hole formation and the relation to transport
Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: Hole formation and the relation to transport
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DOI：
10.1021/bc049962b
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发表时间：
2004-07-01
影响因子：
4.7
通讯作者：
Holl, MMB
中科院分区：
文献类型：
--
作者：
Hong, SP;Bielinska, AU;Holl, MMB
我们使用原子力显微镜（AFM）、酶分析、流式细胞术和荧光显微镜研究了聚氨基胺（PAMAM）树状大分子与负载的1,2二myristoyl- n-glycero-3-phosphocholine （DMPQ）脂质双层和KB和Rat2细胞膜的相互作用。氨基端第7代（G7） PAMAM树状大分子（10-100 nM）在水负载脂质双分子层中形成直径15-40 nM的孔。G5胺端枝状大分子在现有缺陷处没有形成孔洞，反而扩大了孔洞。在此浓度范围内，以乙酰胺为末端的G5 PAMAM树状大分子不会形成空穴。用KB和大鼠2细胞系体外研究PAMAM树突状分子与细胞膜的相互作用。G5胺端和乙酰胺端PAMAM树状大分子在500 nM浓度下均不具有细胞毒性。然而，细胞质蛋白乳酸脱氢酶（LDH）和荧光素酶（Luc）的剂量依赖性释放表明，氨基端G5 PAMAM树突状物的存在降低了细胞膜的完整性。相反，在500 nM浓度下，以乙酰胺为端粒的G5 PAMAM树状大分子的存在对膜的完整性几乎没有影响。胺端枝状大分子引起的通透性诱导不是永久性的，在去除这些树状大分子后，胞质酶的泄漏恢复到正常水平。利用荧光显微镜、LDH和Luc检测以及流式细胞术研究PAMAM树状大分子改变细胞的机制。本研究发现：(1)孔的形成机制与树突内化的观察结果一致；(2)细胞质蛋白可以通过这些孔向细胞外扩散；(3)可以检测到染料分子通过相同的膜孔向细胞内扩散或向细胞外扩散。树状大分子通过孔的扩散足以解释G5胺端PAMAM树状大分子进入细胞的摄取，并且与G5乙酰酰胺端PAMAM树状大分子缺乏摄取是一致的。
We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPQ lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine-nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.