Intracellular Delivery Considerations for RNAi Therapeutics - RNA Interference from Biology to Therapeutics - page 84

In addition to avoiding lysosomal degradation, finding alternatives to endocytosis
as means of internalizing RNAi therapeutics might be desirable also because of the
presence of Toll-like receptors TLR 7 and TLR 8 in the endosomes, which recog-
nize exogenous siRNA and potentiate innate immune response [ 70, 71 ] . Chemical
modi fi cation (i.e., 2 ¢ - O -methyl) of the 2¢-ribose site of siRNA, however, has been
shown to block TLR-mediated immune activation [ 29, 30, 70, 72 ] .
Endosomal escape is one of the most significant barriers encountered during
delivery of RNAi therapeutics, and multiple strategies have been devised that
enhance the efficiency of endosomal escape.

4.3.2.1 Proton Sponge Effect

So-called proton sponge polycations, i.e., polycations with high buffering capacity
in the endo/lysosomal pH range, represent the most widely employed design prin-
ciple in the development of nucleic acid delivery vectors. These polycations are
thought to enhance endosomolysis by absorbing protons and preventing the
acidification of the endosomes, ultimately resulting in the release of the delivery
vectors into the cytoplasm. The proposed mechanism of action relies on elevated
influx of the protons to endosomes mediated by the H+-ATPase. The increased con-
centration of the protons and the counter ions in the endosomal vesicles lead to
increasing osmotic pressure, causing endosomal swelling, membrane rupture, and
finally the release of the RNAi vectors into the cytosol [ 73 ] . Despite growing evi-
dence questioning the validity of the proton sponge hypothesis, polycations that
were designed following this hypothesis, nevertheless, have become among the
most successful ones in nucleic acid delivery.
Polyethyleneimine (PEI) was the first and remains the most wildly used polyca-
tion based on the proton sponge hypothesis [ 31- 33 ]. The mix of primary, secondary,
and tertiary amines in the PEI chain not only provide multiple positive charges at
physiological conditions to bind with the negatively charged siRNA but they also
act as the proton sponge to facilitate the endosomal escape [ 34 ] . Unfortunately, the
high cationic charge density also contributes to high cytotoxicity of PEI, which has
impeded its further applications in vivo [ 74, 75 ]. Efforts have been made to improve
this “gold standard” polycation by conjugating with PEG or by introducing biode-
gradable moieties into the structure to decrease the toxicity [ 35, 36 ] .
PAMAM dendrimers are another group of RNAi delivery vectors with high buff-
ering capacity [ 37- 39 ]. These treelike polymers have highly controlled and defined
sizes and architecture with surface primary amines and central tertiary amines.
These multiple amino groups provide high buffering capacity and proton absorbing
ability, which improve the endosomal escape of the vectors [ 40 ] .

4.3.2.2 Stimuli-Responsive Vectors

Additional approaches to improving the endosomal escape of RNAi therapeutics
rely on stimulus-responsive lipids and polymers. The main categories include pH-
sensitive vectors, temperature-sensitive vectors, and photosensitive vectors [ 76 ] .