Cell-penetrating TAT peptide in drug delivery systems: proteolytic stability requirements

Abstract

The stability and activity of the HIV cell-penetrating TAT peptide (TATp) on the surface of TATp-modified micelles and liposomes in relation to its proteolytic cleavage was investigated. TATp moieties were attached to the surface of these nanocarriers using TATp modified with a conjugate of phosphatidyl ethanolamine with a 'short' PEG (PEG-PE). Following pre-incubation with trypsin, elastase, or collagenase, the proteolytic stability of TATp on the surface of these modified carriers was studied by HPLC with fluorescence detection using fluorenylmethyl chloroformate (FMOC) labeling. All tested enzymes produced a dose-dependent cleavage of TATp as shown by the presence of TATp Arg-Arg fragments. Inhibition of TATp cleavage occurred when these TATp-micelles were modified by the addition of longer PEG-PE blocks, indicating an effective shielding of TATp from proteolysis by these blocks. TATp-modified carriers were also tested for their ability to accumulate in EL-4, HeLa, and B16-F10 cells. Trypsin treatment of TATp-modified liposomes and micelles resulted in decreased uptake and cell interaction, as measured by fluorescence microscopy and fluorescence-activated cell sorting techniques. Furthermore, a decrease in the cytotoxicity of TATp-modified liposomes loaded with doxorubicin (Doxil) was observed following trypsin treatment. In conclusion, steric shielding of TATp is essential to ensure its in vivo therapeutic function.

Figure 1

HPLC TATp RR fragment analysis of enzymatic cleavage following incubation of TATp-modified micelles/liposomes with trypsin/ trypsin inhibitor (0.1mg/ml and 1mg/ml respectively). Particles were incubated with enzymes for 10 min in PBS, pH 7.4, at 37°C before HPLC analysis. (n=3)

Figure 2

PEG shielding of TATp-micelles. HPLC TATp RR fragments analysis of TATp-modified micelles, shielded with increasing Mol% of long PEG2k-PE or PEG5k-PE blocks. TATp enzymatic cleavage was measured following incubation of TATp-modified micelles with trypsin (0.1mg/ml) and presented as percent of cleavage. Particles were incubated with enzyme for 10 min in PBS, pH 7.4, at 37°C. (n=3)

Figure 3

Fluorescence microscopy showing the internalization of rhodamine-PE-labeled TATp-containing liposomes (Red-rhodamine; Blue-Hoechst nuclei staining) by B16-F10, EL4 and HeLa cells following incubation with trypsin (0.1mg/ml) for 1hr at 370C. Both TATp-modified liposomes and TATp-modified liposomes pre-treated with trypsin and with trypsin inhibitor (1mg/ml) enhanced internalization by cells.

Figure 4

Flow cytometry. Average fluorescence intensity of B16-F10, EL4 and HeLa cells incubated with rhodamine-labeled TATp liposomes following exposure to trypsin/trypsin inhibitor (0.1mg/ml and 1mg/ml respectively) for 1hr. (*p≤0.05 vs. plain liposomes, n=5)

Figure 5

In vitro cytotoxicity of a TATp-Doxil formulation towards B16-F10 and HeLa cells. The formulation was pre-treated with 0.1mg/ml trypsin for 1 hr. followed by 24 hr. incubation with cells at 370C before the viability assay. (*p≤0.05 vs. TATp-Doxil, n=5)