Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.
Gene delivery using calcium phosphate nanoparticles: Optimization of the transfection process and the effects of citrate and poly(l-lysine) as additives.
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DOI：
10.1016/j.jcis.2016.03.007
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发表时间：
2016-06-01
影响因子：
9.9
通讯作者：
Uskoković V
中科院分区：
文献类型：
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作者：
Khan MA;Wu VM;Ghosh S;Uskoković V
尽管纳米磷酸钙(CAP)作为一种非病毒转染剂的历史很长，但在优化其性质以进行与病毒载体相当的效率方面取得的成功有限。在这里，我们着重于优化：(A)CAP纳米粒子的沉淀条件，主要是过饱和度和Ca/P摩尔比；(B)转染条件，主要是载体和质粒DNA的浓度；(C)表面添加剂的存在，包括柠檬酸根离子和阳离子聚L赖氨酸(PLL)的存在。与商业非病毒载体相比，CAP纳米颗粒显著提高了编码增强型绿色荧光蛋白(EGFP)的质粒DNA在成骨前MC3T3-E1细胞中的转染率。同时，它们的细胞毒性明显低于商业载体。质粒DNA作为成核促进剂，缩短了亚稳CAP溶液的成核滞后时间，使成核速率较高，析出粒子尺寸较小。过饱和度(DS)15比12.5或17.5或更高的过饱和度更适合于转染法。由于在DS 15处析出的帽状颗粒为球形，而DS 17.5和DS 21处为针状颗粒，因此，球形颗粒比各向异性颗粒更有利于转染体的形成。尽管DS 15的产率分别比DS 17.5和21低10倍和100倍，但使用DS 15制备的帽纳米颗粒胶体的转染率高于在高DS或低DS制备的CAP纳米颗粒胶体，这表明正确的颗粒形态可以盖过载体数量的差异，即使这种差异接近100倍。与商业载体不同，CAP-PDNA进入细胞的浓度与转染率成正比。在骨肉瘤K7M2细胞中，CAP纳米颗粒的转染率是MC3T3-E1细胞的四倍。柠檬酸盐的加入在较低浓度下提高了转染率；然而，在较高浓度的柠檬酸盐下，CAP-PDNA纳米颗粒完全再分散，同时完全减少了转染率，这意味着携带PDNA的CAP纳米颗粒部分聚集的好处。相反，PLL最初延迟了转染率，但在较长的时间点(≥96h)增强了转染率，从而得出结论：柠檬酸盐和PLL都能对转染率产生积极的影响：低浓度的柠檬酸和PLL延长转染期。
Despite the long history of nanoparticulate calcium phosphate (CaP) as a non-viral transfection agent, there has been limited success in attempts to optimize its properties for transfection comparable in efficiency to that of viral vectors. Here we focus on the optimization of: (a) CaP nanoparticle precipitation conditions, predominantly supersaturation and Ca/P molar ratios; (b) transfection conditions, mainly the concentrations of the carrier and plasmid DNA; (c) the presence of surface additives, including citrate anion and cationic poly(L-lysine) (PLL). CaP nanoparticles significantly improved transfection with plasmid DNA encoding enhanced green fluorescent protein (eGFP) in pre-osteoblastic MC3T3-E1 cells compared to a commercial non-viral carrier. At the same time they elicited significantly lesser cytotoxicity than the commercial carrier. Plasmid DNA acted as a nucleation promoter, decreasing the nucleation lag time of metastable CaP solutions and leading to a higher rate of nucleation and a lower size of the precipitated particles. The degree of supersaturation (DS) of 15 was found to be more optimal for transfection than that of 12.5 or 17.5 and higher. Because CaP particles precipitated at DS 15 were spherical, while DS 17.5 and 21 yielded acicular particles, it was concluded that spherical particle morphologies were more conducive to transfection than the anisotropic ones. Even though the yield at DS 15 was 10 and 100 times lower than that at DS 17.5 and 21, respectively, transfection rates were higher using CaP nanoparticle colloids prepared at DS 15 than using those made at higher or lower DS, indicating that the right particle morphology can outweigh the difference in the amount of the carrier, even when this difference is close to 100x. In contrast to the commercial carrier, the concentration of CaP-pDNA delivered to the cells was directly proportional to the transfection rate. Osteosarcoma K7M2 cells were four times more easily transfectable with CaP nanoparticles than the MC3T3-E1 cells. The addition of citrate increased the transfection rate at lower concentrations; however, a complete redispersal of CaP-pDNA nanoparticles at higher concentrations of citrate coincided with a complete diminishment of transfection, implying the benefits of partial aggregation of CaP nanoparticles carrying pDNA. In contrast, PLL delayed transfection initially, but enhanced it at longer time points (≥ 96 h), leading to the conclusion that both citrate and PLL could exert positive effects on transfection: citrate if added at low concentrations and PLL to extend transfection over longer periods of time.