Non-viral Gene Therapy - Intracellular Delivery: Fundamentals and Applications - page 608

2.2.2 Specific Uptake

Numerous lipoplexes and polyplexes have been designed to promote cellular entry
via receptor-mediated endocytosis, in which a panoply of targeting ligands have
been investigated, including endogenous ligands (such as transferrin and RGD
peptide), carbohydrates (galactose, mannose, lactose and etc.), antibodies (like
antiCD3 and antiEGF), and cell penetrating peptides (such as HIV Tat and pol-
yarginine) (Roth and Sundaram 2004 ; Wong et al. 2007 ).
More often, the targeted lipoplexes enter the cell through the pathway that is
involved for the targeting ligand. A ligand that induces clathrin-mediated endo-
cytosis should likewise induce the same endocytotic pathway when incorporated
into lipoplexes. This point has been demonstrated by Brahimi-Horn et al., based
on Arg-Gly-Asp (RGD) decorated lipoplexes (Colin et al. 2000 ). The integrin
targeting lipoplexes were considered to be internalized via clathrin-coated pits
and vesicles, as observed by TEM. The uptake was inhibited by K + depletion
(which inhibits receptor mediated endocytosis by removing clathrin from the
plasma membrane) and hypertonic medium (which blocks clathrin-coated pit
formation). However, studies by Düzgüneş et al. have showed transferrin targeted
lipoplexes (Tf-lipoplexes) deliver transgenes by endocytosis primarily via a non-
receptor-mediated mechanism (Simoes et al. 1999 ). They proposed that the
Tf-mediated enhancement in transfection of Tf-lipoplexes is mainly by two dif-
ferent mechanisms: (1) by triggering internalization of the Tf-lipoplexes through
both non-coated pit (presumably phagocytosis or pinocytosis) and coated pit
mediated endocytosis (after binding of the lipoplexes to non-specific receptors);
and (2) by promoting intracytoplasmic gene delivery in a pH-dependent manner.
Taking together, these results may imply the exact mechanism involved in the
cellular uptake of targeted lipoplexes is related to both ligand type and the physi-
cochemical characters of lipoplexes.
Polyplexes derivatized with targeting ligands are often internalized by recep-
tor-mediated endocytosis. The successful targeting of polyplexes depends on the
conjugation chemistry, the spacer group between ligand and polyplex, the
strength of ligand-receptor binding, and the number of targeting ligands per
polyplex. The ligand-receptor interaction should not be disrupted by conjuga-
tion. Schaffer and Lauffenburgen have found that for EGF-biotin/avidin-
polylysine conjugates, increasing cross-linker (between the EGF and biotin)
length can improve binding affinity, while the short crosslinkers interfered with
EGF/EGF receptor binding (Schaffer and Lauffenburger 1998 ). There also exists
a balance between the specific ligand-receptor interactions and nonspecific elec-
trostatic binding to the cell surface. The specific targeting was found only within
a narrow window of polymer/DNA ratios near electroneutrality. In addition,
there is typically an optimal ligand valency, due to the saturation of both recep-
tor binding and the internalization machinery (Schaffer and Lauffenburger
1998 ). All the various parameters should be carefully optimized to achieve effi-
cient cell-specific targeting.