TAT peptide and its conjugates: proteolytic stability

Abstract

The proteolytic cleavage of TATp, TATp-PEG(1000)-PE conjugate (TATp-conjugate), and TATp as TATp-conjugate in mixed micelles made of TATp-conjugate and PEG(5000)-PE (2.5% mol of TATp-conjugate, TATp-Mic) were studied by HPLC with fluorescent detection using fluorenylmethyl chloroformate (FMOC) labeling and by MALDI-TOF MS analysis. The cleavage kinetics were analyzed in human blood plasma and in trypsin-containing phosphate buffered saline (PBS), pH 7.4, to simulate the proteolytic activity of human plasma. The trypsinolysis of free TATp, TATp-conjugate, and TATp-Mic revealed that the main initial fragmentation is an endocleavage at the carboxyl terminus resulting in an Arg-Arg (RR) dimer. The trypsinolysis followed pseudo-first-order kinetics. The cleavage of the free TATp was relatively fast with a half-life of a few minutes (t(1/2) ∼ 3.5 min). The TATp-conjugate showed more stability with about a 3-fold increase in half-life (t(1/2) ∼ 10 min). TATp in TATp-Mic was highly protected against proteolysis with an over 100-fold increase in half-life (t(1/2) ∼ 430 min). The shielding of TATp by PEG moieties in the proposed TATp-Mic is of great importance for its potential use as a cell-penetrating moiety for multifunctional "smart" drug delivery systems with detachable PEG.

Figure 1

MS spectra of the purified TATp-PEG1000-PE conjugate. The main peak m/z = 3685.42 was identified as the conjugate (theoretical [M+H]⁺ = 3685.21). The intensities of MS peaks do not represent a quantitative ratio. The relative concentration of TATp-Cys did not exceed 7% as determined by HPLC analysis.

Figure 2

MS spectra of TATp fragments resulting from the incubation of TATp in human plasma at 37 °C. Symbols: * [M + Na]⁺ and ** [M + 2Na − H]⁺. (A) t = 0 min (determined by spiking TATp into plasma, in which the proteolytic activity was completely inhibited). (B) t = 20 min (small peptide fragments were not identified, as they could not be distinguished from the background plasma peptides).

Figure 3

The kinetic of TATp proteolysis in human plasma, described by pseudo-first-order kinetics; y = 94.84e^−0.173t (y = % of residual intact TATp); R² = 0.975.

Figure 4

HPLC chromatogram of TATp following FMOC-Cl derivatization. The three separate peaks represent a different extent of derivatization as identified by MALDI-TOF MS (see Figure 5).

Figure 5

MS identification of the relevant peaks 1–3 (see Figure 4) resulting from FMOC derivatization of TATp. 1, TATp-FMOC and nonderivatized TATp; 2, TATp-2FMOC and traces of TATp-FMOC; 3, TATp-3FMOC and traces of TATp-2FMOC and TATp-4FMOC.

Figure 6

HPLC chromatograms of TATp fragments resulting from the trypsinolysis (1.5 trypsin unit/mL) for 20 min in PBS, pH 7.4, at 37 °C. For the proteolysis of TATp-Mic, 100-fold higher concentration of trypsin was used. Fractions of peaks 1, 2, and 3 were collected and identified by MS. The chromatograms represent free TATp (A), TATp-conjugate (B), TATp-Mic (C).

Figure 7

MS identification of the three main fractions (1–3) resulting from TATp proteolysis: (1) RR-FMOC, (2) KR-FMOC, (3) KR-FMOC. The two separate chromatographic peaks of KR-FMOC might result from the two different positions of FMOC labeling: N-terminal or ε-amino group of lysine.

Figure 8

The kinetics of TATp proteolysis by trypsin (1.5 units/mL) in PBS, pH 7.4, at 37 °C. The kinetics was followed by the HPLC based on two different monitoring methods: 1. (—) By determining the decrease in the concentration of the intact TATp. 2. (---) By determining the increase of the RR-fragment normalized to % of the residual intact TATp using the following equation: % Intact TATp = [A_∞ − A_t/A_∞] × 100; A_∞ = RR concentration at the end of proteolysis; At = RR concentration at time t. The kinetics as determined by both methods fit first-order kinetics (as specified in Figure 2) resulting in a similar half-life (t_1/2 = ca. 3.5 min).

Figure 9

Proteolysis kinetics of different forms of TATp by trypsin (1.5 units/mL) in PBS, pH 7.4, 37 °C, following the RR fragment (as specified in Figure 8). TATp (▲), TATp-conjugate (●), TATp-Mic (■).

Figure 10

The principal scheme for the action of stimuli-sensitive nanocarriers. The surface of the nanocarrier is modified with TATp (or other CPP) via a relatively short PEG spacer. The TATp is shielded with longer PEG chains, which are attached to the nanocarrier surface via pH-sensitive bonds. The whole system is stable in the blood with TATp moieties protected against proteolytic degradation, and the carrier accumulates in the tumor via the EPR effect. Inside the tumor, protective PEG chains are detached from the surface by rapid hydrolysis of pH-sensitive bonds at the lower intratumoral pH; intact TATp becomes exposed and allows TATp-mediated intracellular delivery.