PTD-modified ATTEMPTS system for enhanced asparaginase therapy: a proof-of-concept investigation

Abstract

Macromolecular drugs such as proteins and gene products are presumably the most desirable therapeutic agents due to their unmatched substrate specificity and reaction efficiency. Yet, clinical use of these drugs has met with limited success, primarily due to the impermeable nature of the cell membrane that restricts cellular drug uptake to only small (<600 Da) and hydrophobic molecules. The recent discovery of the protein transduction domain (PTD) membrane-penetrating peptides, such as HIV-TAT, has finally offered the possibility of resolving this cell-membrane barrier for macromolecular drug delivery. Via covalent linkages, these PTD peptides have been shown to ferry the attached macromolecular species across membranes of all cell types, both in vitro and in vivo. Nevertheless, the lack of selectivity for PTD-mediated internalization restricts the application of this cell uptake method in clinical practice, due to concerns of inducing systemic toxicity caused by the carried drugs. Presented herein is a modified version of our previously established "ATTEMPTS" approach in delivery of macromolecular drugs, which integrates the cell-penetrating PTDs into a heparin/protamine-regulated delivery system. In vitro findings using asparaginase (ASNase) as a model macromolecular anti-tumor agent were able to validate the feasibility of this delivery system. The chemically constructed TAT-ASNase conjugates not only were able to translocate into the MOLT-4 cells and elicit the cytotoxic effects, but also this PTD-mediated intracellular drug uptake could be regulated (with on/off control) by the addition of heparin and protamine. This modified ATTEMPTS system therefore presents a new avenue of treatment of various types of cancers and other diseases with macromolecular drugs. In vitro characterization and a preliminary proof-of-concept animal investigation that demonstrates the feasibility of this PTD-mediated ASNase therapeutic system is subsequently described.

Figure 1

Schematic of the heparin/protamine delivery system in modulating TAT-mediated intracellular delivery of macromolecular drug.

Figure 2

Elution of ASNase and TAT-ASNase from a heparin column. A gradient of 0.15 – 2.00 M NaCl solution was used to separate ASNase and TAT-ASNase of various heparin binding affinity.

Figure 3

SDS-PAGE of ASNase and TAT-ASNase. ASNase migrated at its expected MW of 36KD whereas TAT-ASNase migrated at higher MW; suggesting approximately 1 to 5 TAT molecules were conjugated to each ASNase.

Figure 4

Fluorescence microscopy of: (A) FITC-ASNase; (B) FITC-TAT-ASNase; (C) FITC-TAT-ASNase with heparin; and (D) FITC-TAT-ASNase with heparin and protamine. HeLa cells were incubated with the different treatment groups.

Figure 5

Flow cytometry analysis of FITC-TAT-ASNase conjugate. (A) ASNase and TAT-ASNase were FITC-labeled and incubated with MOLT-4 cells. (B) FITC-TAT-ASNase was incubated with heparin or heparin and protamine in MOLT-4 cells.

Figure 6

Effect of washing on cell viability. Cell viability was determined with or without washing with 50% FBS after 2 hours incubation with either control medium (black), ASNase (gray) or TAT-ASNase (no color) at 2 IU per 10⁶ MOLT-4 cells. After washing, TAT-ASNase exhibited greater cytotoxicity than ASNase, indicating TAT-ASNase had transduced into MOLT-4 cells and was capable of maintaining its tumor killing ability.

Figure 7

Survival curve for DBA/2 mouse bearing L5178Y mouse lymphoma cells. Each pool of 700,000 L5178Y cells was incubated with 6 IU of either TAT-ASNase (open triangle) or RPMI-1640 solution (positive control; diamond) for 2 hours, followed by washing 3 times with 50% FBS in RPMI. The DBA/2 mice of 6 weeks old were then injected intraperitoneally with one of these three treated tumor cell samples (700,000 cells per sample). Mouse survival times were recorded.