Targeting of hepatocytes using vector-conjugated liposomes : evaluation of targeting strategies

The need for specific targeting strategies towards hepatocytes stems from the lack of
efficient therapeutic options to treat numerous serious liver diseases. Moreover, various
genetic disorders, such as α1-antitrypsin deficiency and hemophilia A and B, are depending
on an efficient gene delivery to defined cells, such as hepatocytes, preferentially avoiding
viral vectors. Since the asialoglycoprotein receptor is primarily expressed by liver
parenchymal cells, it offers a potential target for a cell specific delivery system.
First, the binding of various vectors was analyzed, using the human hepatocellular carcinoma
cell line HepG2 as an in vitro model. While the uptake of D-galactose as a monomer was
non-specific, the glycoprotein asialofetuin was analyzed as an alternative vector, which
represents the desialated derivative of fetuin, containing multi-antennary galactose-
terminating glycan residues. Next to a pronounced cellular accumulation, the uptake was
markedly inhibited in the presence of an excess of free asialofetuin, indicating specific
endocytosis through the asialoglycoprotein receptor. Therefore, asialofetuin was selected as
an ideal vector for the further development of a drug delivery system targeting liver
parenchymal cells.
Asialofetuin was covalently attached to pegylated liposomes, yielding a highly monodisperse
preparation with a particle size below 100 nm. A subsequently incubation with HepG2 cells
resulted in a specific endocytosis of the vesicles, providing an experimental proof of concept
for targeting hepatocytes in vitro. The delivery and intracellular accumulation in HepG2 cells
were investigated by incorporating various organic dyes and fluorescent semiconductor
nanocrystals, also known as quantum dots, into liposomes. The cellular uptake of
asialofetuin-conjugated liposomes, loaded with quantum dots, resulted in a bright fluorescent
signal, which was impaired by the need for a specific photoactivation prior to fluorescence
analysis. Despite their challenging optical properties, quantum dots are valuable
fluorochromes for further optimization of drug targeting strategies.
Finally, a proof of principle for a hepatocyte specific delivery was provided in vivo, by
intravenously injecting rats with asialofetuin-conjugated and pegylated liposomes, which
were taken up by the liver parenchymal cells. In contrast, accumulation in hepatocytes was
reduced by co-injecting free asialofetuin and conventional liposomes were uniquely engulfed
by Kupffer cells.
Summarized, asialofetuin-conjugated pegylated liposomes represent a novel approach,
combining desialated glycoproteins, which exhibit a high affinity towards the
asialoglycoprotein receptor, with long circulating vesicles, for a specific targeting of liver
parenchymal cells. This concept represents a most promising strategy for a hepatocyte
specific drug delivery system and gives the opportunity for further studies, such as the
isolated utilization of glycans only, to avoid immunogenic reactions.
These targeting strategies can be used to deliver drugs to diseased tissues or organs within
our body. This reflects our interests to modulate the pharmacokinetics of drugs using specific
formulation strategies. Two additional pharmacokinetic investigations of pharmaceutical
relevant substances were published in peer-reviewed journals. One study addresses the risk
of physical drug interactions of ceftriaxone with calcium in human plasma, and the second
one discusses the interaction potential of high doses of resveratrol with various cytochrome
P450 isoenzymes. These studies are presented in the section “Appendix”, to separate them
from the drug targeting approach of hepatocytes using liposomal formulations.