ENGINEERING OF CELL-PENETRATING PEPTIDE-CONJUGATED INTRACELLULAR DELIVERY SYSTEMS - Biologically-Responsive Hybrid Biomaterials - page 219

specific cell or organ by attaching the monoclonal antibody (infarct-specific
antimyosin antibody 2G4 or cancer-specific antinucleosome antibody 2C5) to
their surface via reactive pNP-PEG-PE moieties. Second, these liposomes and
micelles were additionally modified with TATp moieties attached to the surface
of the nanocarrier by using TATp-short PEG-PE derivatives. PEG-PE used for
liposome surface modification or for micelle preparation was made degradable
by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-
PE). Under normal pH values, TATp functions on the surface of nanocarriers
were “shielded” by long protecting PEG chains (pH-degradable PEG 2000 -PE or
PEG 5000 -PE) or by long pNP-PEG-PE moieties used to attach antibodies to the
nanocarrier (non-pH-degradable PEG 3400 -PE or PEG 5000 -PE). At pH 7.5-8.0,
both liposomes and micelles demonstrated high specific binding with antibody
substrates, but very limited internalization by NIH/3T3 or U-87 cells. However,
upon brief incubation (15-to-30 min) at lower pH values (pH 5.0-6.0)
nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-
Hz-PE and acquired the ability to be effectively internalized by cells via TATp
moieties (Figure 5).
Fig. 5. Fluorescence microscopy showing internalization of Rh-PE-labeled-TATp containing
micelles by NIH 3T3 fibroblast cells after incubating micelles at pH 8.0 (A), pH 5.0 (B)* for 30
min and RH-PE-labeled-TATp containing liposomes internalization by U87 MG astrocytoma
using: 9 mol % pH-non-sensitive PEG-PE at pH 7.4 (C), 9 mol % pH-sensitive PEG-Hz-PE after
incubation at pH 5.0 for 20 min (D)*. The cleavable (pH sensitive) PEG-PE-based TATp-
containing liposomes/micelles kept at pH 7.4-8 show only marginal association with cells while
those preincubated for 20-30 min at pH 5.0 demonstrated dramatically enhanced association with
the cells (higher fluorescence), i.e., better accessibility of TATp moieties for cell interaction.*The
pH of these formulations was raised back to pH 7.4 after their incubation at pH 5.0 and prior to
incubation with cell.