Potent tumor targeting drug release system comprising MMP-2 specific peptide fragment with self-assembling characteristics

Abstract

Self-assembling peptides are capable of forming a complex containing a cavity where cytotoxic agents can be wrapped in a self-assembling manner. These complexes are beneficial for improving the pharmacological properties and pharmacokinetics of cytotoxic agents, such as doxorubicin and paclitaxel. In the present study, this self-assembling feature was successfully integrated into a hexapeptide with matrix metalloproteinase (MMP)-2 specific targeting activity, producing a supramolecule possessing controlled drug release characteristics. The MMP-2 specific substrate fragment, PVGLIG, makes this supramolecule disassociate in the presence of MMP-2, and this system is considered to be a powerful tool for the treatment of tumors with high expression of MMP-2 or tumor metastasis. Our findings show that this modified self-assembling peptide with the PVGLIG fragment was able to significantly enhance specificity against HT1080 cells, a tumor cell line with high expression of MMP-2. In addition, residence time of the complex in blood was prolonged since paclitaxel was wrapped into the supramolecule. Our results suggest that the modified MMP-2 specific substrate, SAMTA7, could act as a controlled and sustained drug carrier for treatment of tumors with high expression of MMP-2 and for tumor metastasis.

Keywords: drug targeting system; matrix metalloproteinase; paclitaxel; self-assembly.

Figure 1

HPLC comparison of paclitaxel, SAMTA7, and the SAMTA7-paclitaxel combination mixed in different ratios. Notes: Shown are HPLC spectra for (A) paclitaxel, (B) SAMTA7, and the SAMTA7-paclitaxel combination at a ratio of (C) 1:1, (D) 1:5, and (E) 1:6. Samples were injected into an HPLC C18 column and detected at an ultraviolet wavelength of 214 nm. The retention time of paclitaxel and SAMTA7 was 29.6 minutes (A) and 5.4 minutes (B), respectively. A peak at 13.5 minutes was identified as an increased molar ratio of SAMTA7 and paclitaxel (D). At a molar ratio of 1:6, the peak of paclitaxel disappeared, while the appearance of a peak at 13.5 minutes indicated that SAMTA7 might interact with paclitaxel and form a stable complex (E). Abbreviation: HPLC, high-performance liquid chromatography.

Figure 2

Paclitaxel released from a self-assembling supramolecule in the presence of the MMP-2 enzyme. Various complexes of peptides and paclitaxel (paclitaxel 100 μg) were incubated with MMP-2 (5 μg) in phosphate-buffered saline (pH 7.0) containing 100 μM ZnSO₄ at 37°C for 48 hours. High-performance liquid chromatography-mass spectrometry was then used to monitor the free paclitaxel released. The results indicate that the presence of the MMP-2 enzyme induced the release of paclitaxel from the SAMTA7-paclitaxel complex, compared with other complexes formed by other peptides. The supramolecule formed by SAMTA7 initiated drug release at 2 hours after incubation with MMP-2 and the paclitaxel release from the supramolecules was complete at 24 hours for SAMTA7. It was considered that SAMTA7 formed an MMP-2-sensitive supramolecule containing paclitaxel. Abbreviation: MMP, matrix metalloproteinase.

Figure 3

Evaluation of inhibition of tumor cell metastasis in HT-1080 (A) and HEp2 (B). The SAMTA7-paclitaxel complex had a remarkable antiproliferation effect on HT1080 cells. Notes: HT1080 and HEp2 cells were seeded in 24-well plates and cultured overnight to form a confluent monolayer. After being scratched with a sterile p200 pipette tip, the cell culture containing either paclitaxel or SAMTA7-paclitaxel (final concentration of paclitaxel 5 μg/mL, molar ratio 1:6) was added to scratched plates and incubated at 37°C in a 5% CO₂ atmosphere for 24 hours. The distances between the two edges of the scratched cells were observed using microscopy. The metastasis inhibition rate was calculated by: inhibition rate = (distance of experimental group after inhibition – distance of control group after inhibition – distance prior to inhibition)/distance prior to healing ×100. Compared with the migration inhibiting effect of free paclitaxel in HT1080 cells, the supramolecule showed a 5.3-fold increase in antimigratory activity. However, the SAMTA7-paclitaxel complex showed poor antimigratory activity in HEp2 cells, which do not express MMP-2. It was assumed that the inhibitory effect of the supramolecule on the cellular migrating phenotype is due to the specific release of paclitaxel in the presence of MMP-2 enzyme. Abbreviations: PBS, phosphate-buffered saline; MMP, matrix metalloproteinase.

Figure 4

Effect of the SAMTA7-paclitaxel complex on human xenograft tumor model mice bearing (A) HT1080 cells, (B) HEp2 cells, and (C) U-87MG cells. (D) Antitumor effect of the SAMTA7-paclitaxel complex in tumor-bearing mice. Notes: Mice bearing HT1080, HEp2, and U-87MG xenografts were treated with the supramolecule and with free paclitaxel. The antitumor efficacy of the SAMTA7-paclitaxel supramolecule was measured by monitoring the tumor volume after treatment of tumor-bearing mice. Survival time was recorded in days after tumor injection. All data obtained for repeated experiments were pooled and used for the statistical analysis. The results indicate that tumor size decreased more in mice treated with the SAMTA7-paclitaxel complex than in HT1080-bearing mice treated with free paclitaxel. However, the supramolecule exhibited poor antitumor activity in mice bearing HEp2 and U-87MG tumors. In addition, the group treated with the SAMTA7/paclitaxel complex (•) exhibited efficient antitumor activity as shown. Experimental animals showed an improved survival time compared with those treated with free paclitaxel (□; P<0.05, n=10). Abbreviation: PBS, phosphate-buffered saline.

Figure 5

Computational simulation of SAMTA7 (A) and the SAMTA7-paclitaxel complex (B). The initial system containing SAMTA7 peptides was constructed using the Molecular Operating Environment system. Paclitaxel and six SAMTA7 molecules were immersed into an anhydrous simulated box for MD simulations simultaneously which were performed for 10 msec for the anhydrous system. The time step was 1 fsec, and the trajectories were saved every 20 psec for further analysis. A GROMOS96 53a6 force field was used. The reference temperature was set to 310 K. Simulation of a single molecule of SAMTA7 showed that this peptide was remained in a “hairpin-like” conformation upon interaction from RADA fragments. This conformation explored the MMP-2 substrate peptide, PVGLIG fragment, explaining how SAMTA7 is vulnerable to MMP-2 proteolysis. In (B), six SAMTA7 molecules and one paclitaxel molecule were immersed into the simulation box for structural analysis of the supramolecule, data indicating that SMATA7 was capable of wrapping the free paclitaxel in cavity in self-assembling manner. Importantly, formation of the supramolecule did not alter the phenomenon whereby PVGLIG fragments remained exposed against MMP-2 protein. Abbreviation: MMP, matrix metalloproteinase.