Influence of "Flexible" versus "Rigid" Nanoparticles on the Stability of Matrix Metalloproteinase-7

Abstract

Matrix Metalloproteinase-7 (MMP-7) is invariably expressed in a variety of cancer cells, and exhibits the potentials to interact with differently charged macromolecular surfaces 1. To ascertain whether the nature of the charge carrying surfaces influences the stability as well as catalytic properties of the enzyme, we compared the effects of differently charged lipid (representative of "flexible") and gold ("rigid") nanoparticles. The experimental data revealed that the catalytic activity of MMP-7 is impaired only by the positively charged lipid nanoparticles, and it remains unaffected by their negatively charged or neutral counterparts. On the other hand, both positively and negatively charged gold nanoparticles impair the enzyme activity with nearly equal potency; no significant influence of neutral gold nanoparticles was noted on the enzyme activity. Unlike lipid nanoparticles, the charged gold nanoparticles mediated effects were found to be manifested partially via the inactivation of the enzyme. Arguments are presented that both the "rigidity" as well as the surface curvature of the lipid ("flexible") vis a vis the gold ("rigid") nanoparticles are responsible for eliciting differential influence on the catalytic activity as well as the stability of MMP-7.

Figure 1

Effect of differently charged LNPs on MMP-7 catalyzed reaction. The time course of the MMP-7 catalyzed reaction was monitored (Ex = 335 nm, Em = 395 nm) using the fluorogenic peptide as a function of increasing concentrations of differently charged LNPs in 25 mM HEPES, 10 mM CaCl₂, pH 7.5. The symbol •-•, ○- ○, and □- □ represent data obtained in presence of cationic, anionic and neutral LNPs respectively. [MMP-7] = 0.3 μM, [Substrate] = 10 μM at pH 7.5. Note unlike anionic and neutral LNPs, cationic LNP inhibits the MMP-7 catalyzed reaction. The solid smooth line is the best fit of the data according to Banerjee et al . The K_i value of the cationic LNP-MMP-7 complex complex was found to be 18.5 μM. For the anionic and neutral LNPs, the dashed line represents the trends of the data points.

Figure 2

The effect of differently charged AuNPs on MMP-7 catalyzed reaction. Other experimental conditions are the same as in Figure 1. The symbol •-•, ○- ○, and □- □ are cationic, anionic and neutral AuNPs respectively. [MMP-7] = 0.3 μM, [Substrate] = 10 μM. Note that Au-CNP and Au-ANP (but not the Au-NNP) result in decrease in the MMP-7 catalyzed reaction. The solid smooth line is the best fit of the data for the anionic and cationic AuNPs. IC₅₀ values for the Au-CNP and Au-ANP dependent inhibition data were obtained by fitting them by Eq 1 (see Results and Discussion), and its value was found to be 0.178 ± 0.030 μM. For Au-NNP, the solid line represents the trends of the data points.

Figure 3

Inhibition versus inactivation of MMP-7 by Au-CNP. The time course of the MMP-7 catalyzed reaction in the presence of Au-CNP is shown. Control experiment has been performed in the absence of Au-CNP. The time course of the reaction was monitored for about 1000 sec. Other conditions are same as in Figure 2. The black solid line, the red solid line and the blue solid line indicates the progress curves of MMP-7 alone, MMP-7 with 0.050 μM Au-CNP (0 time incubation) and MMP-7 (10 min pre-incubation time) respectively. The slopes (ΔF s⁻¹⁾ of the progress curves were calculated for the above data were 0.38 (black line), 0.28 (red line), and 0.087 (blue line), respectively. Note that the incubation of MMP-7 with Au-CNP for 10 min resulted in an additional 70% loss in the enzyme activity.

Figure 4

Circular dichroic spectra of MMP-7 in the presence of increasing concentration of Au-CNP. The experiment was performed in 5 mM phosphate buffer, pH 7.5 between 210 – 270 nm region using Jasco J-815 spectropolarimeter. The concentration of Au-CNP varied increased from 0 to 5 μM. The solid green line indicates the CD spectra of 50 μM MMP-7 alone. The increasing concentration of Au-CNP results in the decrease in the magnitude of the negative ellipticity as shown by the vertical arrow.

Figure 5

Cartoon showing the influence of “flexible” versus “rigid” cationic surface on the structural feature of MMP-7.