Biomedical Engineering Reference
In-Depth Information
3.2
TATp- Modified Liposomes for Delivery of Nucleic Acids
Another exceptionally important yet challenging task is cellular delivery of nucleic
acids. Various methods used to deliver these highly negatively charged biomolecules
are associated with cellular toxicity or poor efficiency in certain types of cells
(e.g. lipofectamnine or microinjection). Currently, liposomes and cationic polymers
are used for transfection but they are also less efficient and often accompanied by
high levels of toxicity.
Due to the size of plasmids and the high number of negative charges, non-
covalent approach have been mostly used. It has been reported that TATp binds to
DNA to form complexes which can be internalized through endocytosis (Sandgren
et al.
2002
). In our laboratory we prepared TATp-liposomes with the addition of a
small quantity of a cationic lipid (DOTAP) and incubated with DNA to form stable
non-covalent complexes with a model gene encoding for the enhanced-green fluo-
rescent protein (pEGFP-N1) (Torchilin and Levchenko
2003
). Such TATp-
liposome-DNA complexes when incubated with mouse fibroblast NIH 3T3 and
cardiac myocytes H9C2 showed substantially higher transfection
in vitro
, with
lower cytotoxicity than the commonly used Lipofectin
®
.
We have also investigated the potential of TATp-modified liposomes to
enhance the delivery of the model gene, pEGFP, to human brain tumor U-87 MG
cells
in vitro
and in an intracranial tumor model in nude mice (Gupta et al.
2007
). The size distribution of DNA-loaded TATp-liposomes was narrow
(around 250 nm) and the DNA complexation was firm at lipid/DNA (+/−) charge
ratios of 5 and higher. TATp-lipoplexes demonstrated an enhanced delivery of
pEGFP to U-87 MG tumor cells
in vitro
at lipid/DNA (+/−) charge ratios of
5 and 10.
In vivo
transfection of intracranial brain tumors by intratumoral injec-
tions of TATp-lipoplexes showed an enhanced delivery of pEGFP selectively to
tumor cells and subsequent effective transfection compared to plain plasmid-
loaded lipoplexes. No transfection was observed in the normal brain adjacent to
the tumor. Thus, TATp-lipoplexes can be used to augment the delivery of genes
to tumor cells when injected intratumorally, without affecting the normal adja-
cent brain.
Another example is gene delivery into immunocompetent cells to modulate
immune response. Antigen presenting cells (APC) are among the most important
cells of the immune system since they link the innate and the adaptive immune
responses, directing the type of immune response to be elicited. However, APC
are very resistant to transfection. To increase the efficiency of APC transfection,
we used liposome-based lipoplexes additionally modified with TATp for better
intracellular delivery of a model pEGFP. pEGFP-bearing lipoplexes made of a
mixture of egg phosphatidylcholine (PC): cholesterol (Chol): DOTAP (60:30:10 M
ratio) with the addition of 2% mol of PEG-PE conjugate (plain-L) or TATp-
PEG-PE (TATp-L) effectively protected the incorporated DNA from degradation.
Uptake assays of rhodamine (rh)-labeled lipoplexes and transfections with the
EGFP reporter gene were performed with APC derived from the mouse spleen.
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