Lipid nanoparticles assessment by flow cytometry.
BACKGROUND
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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Institute of Chemical Technology and Engineering, Poznan University of Technology, 4 Berdychowo Street, 60-965 Poznan, Poland. Electronic address: anna.bryla@doctorate.put.poznan.pl.
Anna
A
Bryła
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 48 Wojska Polskiego Street, 60-627 Poznan, Poland. Electronic address: w_juzwa@up.poznan.pl.
Wojciech
W
Juzwa
Institute of Physics, Poznan University of Technology, 3 Piotrowo Street, 60-965 Poznan, Poland. Electronic address: marek.l.weiss@doctorate.put.poznan.pl.
Marek
M
Weiss
Department of Biotechnology and Food Microbiology, Poznan University of Life Sciences, 48 Wojska Polskiego Street, 60-627 Poznan, Poland. Electronic address: gralew@up.poznan.pl.
Grażyna
G
Lewandowicz
D000067493:Dynamic Light Scattering; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008081:Liposomes / Q000032:analysis* / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D053758:Nanoparticles / Q000032:analysis* / Q000737:chemistry*; D010316:Particle Size
D005456:Fluorescent Dyes; D008081:Liposomes
Drug carriers; Drug delivery; Flow cytometry; Liposomes; Nanocarriers