Mass Transport via Cellular Barriers and Endocytosis - Intracellular Delivery: Fundamentals and Applications - page 31

direct vesicle escape) are an important focus of nano-medical research. Recent
research has shown that these objectives can be accomplished. For example, nano-
particles have been utilized to deliver siRNA (Chen et al. 2009 ), RNase A (Bale
et al. 2010 ), Cyt C (Slowing et al. 2007 ; Park et al. 2010a ) and plasmids into the
cytoplasm, from where nucleic core complexes direct the plasmids into the nucleus
(Tros de Ilarduya et al. 2010 ). With the exception of a select few particles which
directly penetrate the cellular membrane in order to reach the cytoplasm, a nano-
particle must be capable of escaping the degradative environment of the late-
endosomes and lysosomes which serve three main objectives: (1) to digest and store
metabolic material for the cell, (2) to digest and recycle aged proteins and organ-
elles, and (3) to protect the cell from engulfed external agents. In order for these
organelles to accomplish these three objectives, they must be able to digest their
internalized material and selectively transport desired agents across their intracel-
lular membranes. Though these two functions are distinct, they are closely linked.
To perform a digestive function, the internal environment of organelles must be
closely maintained by their specialized membranes at a low pH. The low pH of
these organelles is optimal for many proteolytic, glycanolytic, lipolytic and
nuclease enzymes. The abundance of protons also serves as a catalyst for degrada-
tive organic reactions (Holtzman 1989, Storrie B ; Ivanov 2008 ). The pH decreases
constantly as material travels from the cellular membrane (in early endosomes) to
the lysosome, and a particle can escape the vesicle at any point along the pathway;
however the two organelles are intrinsically different. In fact, lysosomes are known
to exist as separate entities before merging with endosomes and subsequently
engulfing their contents. In this way, lysosomes are able to conserve energy and
recycle their enzymes rather than expending energy by creating new zymogens,
enzymes, membrane proteins, and lysosomal membrane phospholipids (Holtzman
1989 ; Storrie B ; Ivanov 2008 ).
A variety of membrane transporters for products such as carbohydrates, amino
acids, nucleic acids, enzymes, and ions serve to regulate the hostile environment
inside of late-endosomes and lysosomes. However, two families of membrane
markers and one membrane transporter are of particular interest. The Rab-family
membrane markers distinguish between endosomes (specific species distinguish
between different types) and lysosomes (the lysosomal associated membrane pro-
teins (LAMP)). The membrane transporter of note is the vacuolar proton trans-
porter V+ATPase. This transporter is considered to be the dominant transporter in
the acidification of endosomes to lysosomes.
Since the 1990's, at least four different mechanisms have been proposed for
endosomal or lysosomal escape: the use of pH-sensitive fusogenic peptides,
“fusogenic” lipoplexes, dynamic polyconjugates and proton-sponge polymers or
materials (Paillard et al. 2010 ; Sasaki et al. 2008 ; Zelphati and Szoka 1996 ;
Rozema et al. 2007 ; Akinc et al. 2005 ). Of these four, the latter three utilize the
low pH of the organelle for their escape while the first capitalizes on the properties
of the phospholipid bilayer, which is predominantly zwitterionic on the inside and
anionic on the outside. The “fusogenic” lipoplexes are composed of cationic lipids
complexed around an oligonucleotide core. The cationic lipids not only protect the