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首页> 外文期刊>Biochemistry >Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.
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Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.

机译：CDAN / DOPE和DC-Chol / DOPE脂质复合物的热力学方面和生物学特性。

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The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies showthat DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.
机译：两种已建立的阳离子脂质体制剂CDAN / DOPE（50:50，m / m; Trojene）和DC-Chol / DOPE（60:40，m / m）的DNA络合和缩合特性通过结合使用等温滴定热量法（ITC），圆二色性（CD），光子相关光谱法（PCS）和浊度测定法。用挤出的脂质体（90 +/- 15nm）滴定质粒DNA（7528bp），并建立热力学曲线。 ITC数据显示，这两种脂质体制剂的DNA络合特性大不相同。通过将实验数据拟合为一个获得CDAN / DOPE（K（d）= 19 +/- 3 microM）和DC-Chol / DOPE脂质体（K（d）= 2 +/- 0.5 microM）的平衡解离常数站点绑定模型。 CDAN / DOPE和DC-Chol / DOPE结合事件都是在负的结合焓（分别为DeltaH度= -0.5和-1.7 kcal / mol）和增加的系统熵（TDeltaS = 6 +/- 0.3和6.2 + / -分别为0.3 kcal / mol。有趣的是，CDAN / DOPE脂质体在与pDNA络合之前会经历大量的水化和质子化，这在滴定过程中被观察为两个离散的放热信号。关于DC-Chol / DOPE与pDNA之间的结合，没有看到这样的双相作用，否则似乎是瞬时的，没有补液作用。 ITC证实了CDAN / DOPE脂质体与DC-Chol / DOPE阳离子脂质体相比，其水化和质子化特性。 CDAN / DOPE脂质体具有强烈的放热稀释特性，而DC-Chol / DOPE脂质体仅具有轻度的吸热特性。此外，通过CD光谱法分析阳离子脂质体-pDNA结合发现，CDAN / DOPE-pDNA脂质复合物比DC-Chol / DOPE-pDNA脂质复合物在结构上更具流动性。 CDAN / DOPE脂质体至少在60分钟内引起DNA结构的显着波动，而从DC-Chol / DOPE获得的脂质体对DNA结构缺乏相同的作用。浊度研究表明，DC-Chol / DOPE脂质复合物显示出比CDAN / DOPE脂质复合物更大的血清抗药性，后者与血清孵育100分钟后显示出大量沉淀。 HeLa和Panc-1细胞的转染研究表明，CDAN / DOPE脂质复合物的功效优于DC-Chol / DOPE脂质复合物。 CDAN / DOPE脂质体倾向于在正常生长培养基（包括10％的血清和抗生素）中转染效果最好，而DC-Chol / DOPE脂质体在无血清转染条件下转染效果最好。

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