"SMART" drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers

Abstract

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or 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, biotin and 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 even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 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 become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.

Figure 1

Interaction of the multifunctional pH-responsive pharmaceutical nanocarrier with the target cell. Local stimuli-dependent removal of protecting PEG chains or mAb-PEG moieties allows for the direct interaction of the CPP moiety with the cell membrane.

Figure 2

Schematic for the design of the multifunctional DDS used in this study that includes pH-cleavable PEG-Hz-PE (a), temporarily “shielded” biotin or TATp (b), and monoclonal antibody (c) attached to the surface of DDS via pH-non-cleavable spacer.

Figure 3

Schematic description of the conjugation reaction.

Figure 4

HPLC analysis of the pH-sensitive mPEG₂₀₀₀-Hz-PE micelles after incubation in pH 8.0 (A) and after incubation in pH 5 (B) at room temperature.

Figure 5

Binding of antimyosin mAb 2G4-PEG₂₀₀₀-Hz-PE-immunomicelles to a monolayer of dog cardiac myosin in comparison to the native mAb 2G4 at corresponding pH values.

Figure 6

Binding of pH-sensitive biotin-micelles to NeutrAvidin columns after 15 min incubation at room temperature at pH 8.0 (a) and at pH 5.0 (b).

Figure 7

Fluorescence microscopy showing internalization of Rh-PE-labeled-TATp containing micelles by NIH 3T3 fibroblast cells after incubating micelles at pH 8.0 (A) and pH 5.0 (B) for 30 min.

Figure 8

Fluorescence microscopy showing internalization of Rh-PE-labeled-TATp containing liposomes by U-87 MG astrocytoma cells using: PEG-free TATp-liposomes (A); 9 % mol pH-non-cleavable PEG-PE at pH 7.4 (B); 18 % mol pH-non-cleavable PEG-PE at pH 7.4 (C); 9 % mol pH-cleavable PEG-Hz-PE after incubation at pH 5.0 for 20 min (D)^§, 18 % mol pH-cleavable PEG-Hz-PE after incubation at pH 5.0 for 20 min (E)^{§. §}The pH of these formulations was raised back to pH 7.4 after their incubation at pH 5.0 and prior to incubation with cells.