Institute of Chemical Technology and Engineering, Poznan University of Technology, 4 Berdychowo Street, 60-965 Poznan, Poland.
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 48 Wojska Polskiego Street, 60-627 Poznan, Poland.
Int J Pharm. 2017 Mar 30;520(1-2):149-157. doi: 10.1016/j.ijpharm.2017.01.047. Epub 2017 Feb 1.
Liposomes are promising carriers for drugs and bioactive compounds. Size and structure are their crucial parameters. Thus, it is essential to assess individual vesicles as prepared. Currently available techniques fail to measure liposome's size and structure simultaneously, with a high throughput. To solve this problem, we have developed a novel, flow cytometric method quantifying liposomes.
Firstly, the following fluorescent staining combinations were tested: DiD/TO, Rh123/DiD, Syto9/DiD. Further, chosen fluorochromes were used to compare three populations of vesicles: raw (R), obtained by thin film hydration and extruded ones (populations E10 and E21). Dynamic light scattering (DLS) was used for determination of average diameter and size distribution of nanocarriers. Structural differences between the raw and the extruded liposomes, as well as additional information concerning vesicles size were acquired employing atomic force microscopy (AFM).
DLS analysis indicated that, three distinct populations of vesicles were obtained. Liposomes were characterized by mean diameter of 323nm, 220nm and 170nm for population R, E10 and E21 respectively. All the populations were stable and revealed zeta potential of -29mV. AFM confirmed that raw and extruded liposomes were differed in structure.
DiD/TO was the optimal fluorochrome combination that enabled to resolve distinctly the sub-populations of liposomes. Results obtained by flow cytometry were in a good agreement with those from DLS and AFM. It was proved that, flow cytometry, when proper fluorescent dyes are used, is an adequate method for liposomes assessment. The proposed method enables fast and reliable analysis of liposomes in their native environment.
脂质体是药物和生物活性化合物的有前途的载体。大小和结构是它们的关键参数。因此，评估制备的单个囊泡至关重要。目前可用的技术无法同时以高通量测量脂质体的大小和结构。为了解决这个问题，我们开发了一种新型的、流式细胞术定量脂质体的方法。
首先，测试了以下荧光染色组合：DiD/TO、Rh123/DiD、Syto9/DiD。此外，选择的荧光染料用于比较三种囊泡群体：原始（R）、通过薄膜水化和挤出获得的囊泡（E10 和 E21 群体）。动态光散射（DLS）用于确定纳米载体的平均直径和粒径分布。使用原子力显微镜（AFM）获得原始和挤出脂质体之间的结构差异以及有关囊泡大小的其他信息。
DLS 分析表明，获得了三种不同的囊泡群体。群体 R、E10 和 E21 的平均直径分别为 323nm、220nm 和 170nm。所有群体均稳定，zeta 电位为-29mV。AFM 证实原始和挤出的脂质体在结构上有所不同。
DiD/TO 是最佳的荧光染料组合，可以清楚地区分脂质体的亚群体。流式细胞术获得的结果与 DLS 和 AFM 的结果非常吻合。事实证明，当使用适当的荧光染料时，流式细胞术是评估脂质体的一种合适方法。所提出的方法能够快速可靠地分析其天然环境中的脂质体。
