Moghimi S Moein, Hamad Islam, Andresen Thomas L, Jørgensen Kent, Szebeni Janos
Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Cockcroft Bldg., Lewes Rd., Brighton BN2 4GJ, UK.
FASEB J. 2006 Dec;20(14):2591-3. doi: 10.1096/fj.06-6186fje. Epub 2006 Oct 25.
Methoxy(polyethylene glycol), mPEG, -grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non-IgE-mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid-mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid-mPEG conjugate responsible for PEGylated liposome-induced complement activation in normal as well as C1q-deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid-mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid-mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b-9, C4d, and C3a-desarg levels in normal human sera as well as SC5b-9 in EGTA-chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid-mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated phospholipid-mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid-mPEG micelles had no effect on complement activation, thus suggesting a possible role for vesicular zwitterionic phospholipid head-groups as an additional factor contributing to PEGylated liposome-mediated complement activation. Our findings provide a rational conceptual basis for development of safer vesicles for site-specific drug delivery and controlled release at pathological sites.
甲氧基（聚乙二醇），即mPEG，接枝的脂质体在血液中具有延长的循环时间，但将其注入相当比例的人体受试者会引发即时的非IgE介导的超敏反应。人们坚信这些反应是由补体激活产生过敏毒素所致。尽管普遍认为用mPEG对囊泡表面进行伪装应能显著抑制补体激活，但我们在此表明，用磷脂 - mPEG共轭物对非补体激活脂质体（二棕榈酰磷脂酰胆碱，即DPPC囊泡）进行双层富集可诱导补体激活，导致巨噬细胞补体受体识别囊泡。然而，囊泡摄取的程度取决于表面mPEG密度。我们使用带有经典以及新合成的脂质 - mPEG共轭物的DPPC囊泡，描绘了磷脂 - mPEG共轭物在正常以及C1q缺陷型人血清中导致聚乙二醇化脂质体诱导补体激活的可能结构特征。对于聚乙二醇化的DPPC囊泡，磷脂 - mPEG共轭物磷酸部分的净阴离子电荷在补体经典途径和替代途径的激活以及过敏毒素产生中起关键作用（反映在正常人血清中SC5b - 9、C4d和C3a - desarg水平显著升高以及EGTA螯合/Mg2 +补充血清中SC5b - 9升高），因为磷脂 - mPEG共轭物磷酸氧的甲基化，进而去除负电荷，完全阻止了补体激活。为了进一步证实负电荷在补体激活中的作用，已表明带有阴离子磷脂 - mPEG共轭物而非甲基化磷脂 - mPEG的囊泡可显著降低大鼠血清溶血活性并提高血浆血栓素B2水平。与脂质体相反，磷脂 - mPEG胶束对补体激活没有影响，因此表明囊泡两性离子磷脂头部基团可能作为促成聚乙二醇化脂质体介导补体激活的另一个因素。我们的研究结果为开发用于在病理部位进行位点特异性药物递送和控释的更安全囊泡提供了合理的概念基础。
