| Abstract |
Liposomes are used as drug delivery systems in the past two decades and they
have quite a lot of applications in different fields of science. In acoustic biosensors
liposomes due to their structure are used to determine different characteristics of
liposomes and to find out innovative properties of the bound vesicles. Within this
thesis, the potential of a novel-method for the quantification of surface bound DNAloaded
liposomes has been investigated.
In this work liposomes were produced using the widely known thin film
hydration method. During this process DNA was diluted into the medium and
entrapped into the liposomes. After the extrusion and the formation of DNA-loaded
liposomes, the latter were purified from non-encapsulated DNA via several different
methods, including overnight dialysis and centrifugation. These methods were
compared during this project to determine the most efficient method for removing the
non-entrapped DNA. The encapsulation efficiency of the liposomes has been
determined by quantifying separately the encapsulated DNA and the number of
liposomes produced. The quantification of the encapsulated DNA via qPCR
measurement along with the lipid quantification provided the basis for the calculation
of the DNA/liposome ratio. This ratio was used for the quantification of bound
liposomes during the acoustic experiments.
The acoustic biosensor used was the Quartz Crystal Microbalance with
Dissipation monitoring (QCM-D). In this work, liposomes containing 1% of
biotinylated lipids were captured on the sensor surface using the biotin-neutravidin
capture system. Liposomes are bound on the previously Neutravidin-coated biosensor
surface via their biotinylated lipids, changing the acoustic wave propagation
characteristics. In order to quantify the liposomes bound, the liposomes were
disrupted using detergents and the waste coming out of the sensor chambers was
collected and analyzed. For this purpose, two different methods were used and
compared. In the first one, the quantification was succeeded indirectly, via qPCR
experiments and via the DNA/liposome ratio. In the second method, the number of
liposomes previously bound on the surface was determined directly by
Phosphatidylcholine quantification (PC hydrolysis). Finally, a correlation between the
number of bound liposomes calculated from the two different methods and the signal
from QCM-D was performed.
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A novel method for the quantification of surface bound DNA- loaded liposomes
