Biomedical Engineering Reference
In-Depth Information
while DOTAP/cholesterol (1:1 w:w) showed approximately 5% and
8% transfection, respectively. Therefore, small unilamellar cationic
liposomes are prepared with DOTAP and cholesterol in 1:1 molar
ratio by thin film hydration followed by membrane extrusion for the
LPD formulation.
Several research groups have reported that siRNA interacts
with cationic carriers with less strength than the plasmid DNA,
resulting in the formation of unstable particles and reduced
delivery efficiency.
92, 93
Self-assembly of macromolecules by charge
interaction is largely driven by the loss of the bound counterions
and is hence an entropy driven process.
94
Nucleic acids less than
140 nucleotides long are not readily condensed by polycations.
Typical siRNA is only 19-22 nucleotides in length and cannot by
itself be stably condensed by a polycation, such as protamine. In the
case of LPD, an appropriate amount of calf thymus (about 50 kbp)
or plasmid DNA (about 6 kbp) was mixed with siRNA to promote
the stability of the condensed NPs.
92
The amount of polycationic
carriers can be varied according to the nucleic acid cargo of interest.
Despite the fact that the plasmid DNA can form a tighter complex
with cationic carriers, calf thymus DNA has an advantage as carrier
DNA due to its limited amount of immune stimulating CpG motif.
Because of the high molecular weight, the LPD particle size can be
reduced by 10-30% and the delivery efficiency may be increased by
20-80% when calf thymus DNA is used as carrier DNA.
95
After the
mixture of nucleic acid and carrier DNA is prepared (1:1, w:w), a
highly positively charged peptide, protamine, may be added to the
mixture for further condensation of the negatively charged DNA
complex. The delivery efficiency of NPs increases with increasing
protamine concentration. However, the net charge of the protamine-
DNA complex becomes slightly positive when excess of protamine is
added, which eliminates the interaction with the cationic liposome,
and results in a reduction of the delivery efficiency. Moreover,
the amount of protamine should be adjusted according to the
conformation of nucleic acids, as the double stranded siRNA needs
more protamines for core condensation than the single stranded AS-
ODN. The last step of the self-assembly process is the insertion of
PEG-lipid conjugate to the LPD to prevent interaction with serum
proteins. The distal end of the PEG chain is tethered with a ligand,
such as anisamide, to promote nanoparticle interaction with tumor
cells that over-express the sigma receptor. The entire process of self-
assembly is schematically shown in Fig. 3.3.
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