Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2 S Donor Conjugate with Complexed Fe2

Abstract

Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe²⁺ , CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H₂ S donor conjugate, complexed with Fe²⁺ , termed AAN-PTC-Fe²⁺ . The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H₂ S, an inhibitor of catalase, an enzyme that detoxifies H₂ O₂ . Fe²⁺ and H₂ S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe²⁺ , the AAN sequence, or the ability to generate H₂ S. AAN-PTC-Fe²⁺ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H₂ S-amplified, enzyme-responsive platform for synergistic cancer treatment.

Keywords: Cancer; Drug Delivery; Gasotransmitter; Iron; Prodrugs.

Figure 1.

Schematic illustration of PHDC–Fe²⁺ complex chemical structure and therapeutic mechanism in C6 glioma cell line.

Figure 2.

Chemical structures of control PHDCs: A. AAQ-PTC, which does not respond to legumain, and thus cannot release H₂S. B. AAN-PC, which responds to legumain but releases CO₂ instead of COS, and thus cannot generate H₂S.

Figure 3.

A. Representative H₂S release profiles of AAN-PTC, AAQ-PTC, and AAN-PC (333 μM) triggered by CA (3.33 μM) and legumain (1 ng/μL) at rt in acetate buffer (10 mM, pH 5.0). Data were obtained on an H₂S-sensitive electrochemical probe. B. H₂S release kinetics from AAN-PTC, AAQ-PTC, and AAN-PC (100 μM) measured via the methylene blue assay in acetate buffer (10 mM, pH 5.0) with CA (1 μM) and legumain (1 ng/μL). Data points are solid circles, and the solid line shows the pseudo-first-order kinetics fit for AAN-PTC. Error bars indicate standard deviations of three separate experiments.

Figure 4.

A. Brightfield, fluorescence, and merged images showing fluorescence in C6 cells preincubated with H₂S probe WSP-5 (50 μM) for 30 min and then treated with the following groups for 3 h: blank (1× PBS), Na₂S (200 μM), AAN-PTC (with cleavable site and H₂S donor, 200 μM), AAQ-PTC (with H₂S donor but no cleavable site, 200 μM), AAN-PC (with cleavable site but no H₂S donor, 200 μM). Cells were then washed, and fluorescence images were taken in PBS. Scale bars are 100 μm. B. Quantification of corrected total cell fluorescence (CTCF). Average fluorescence intensities were quantified by ImageJ (cell counts are >30 for each group from three separate wells). The error bars represent the standard deviation between repeats (n=3).

Figure 5.

A. ITC thermogram of the AAN-PTC–Fe²⁺ titration. FeCl₂ solution (10 mM) was titrated into AAN-PTC solution (1 mM) at 298 K. B. ITC titration curve of AAN-PTC–Fe²⁺ titration, the red solid line is the nonlinear least-squares fitted curve, and the dashed line at 0.95 indicates the binding stoichiometry (N).

Figure 6.

A. Representative bright field, live/dead, and merged images of C6 glioma cells treated with PBS buffer (blank), FeCl₂ (200 μM), AAN-PTC (with cleavable site and H₂S donor but without Fe²⁺ complexed, 200 μM), AAQ-PTC–Fe²⁺ (with H₂S donor but no cleavable site, 200 μM), AAN-PC–Fe²⁺ (with cleavable site but no H₂S donor, 200 μM), AAN-PTC–Fe²⁺(with cleavable site and H₂S donor, 200 μM), and TMZ (1 mM) for 24 h post-seeding. Live/dead staining (calcein-AM and ethidium-Br) and subsequent fluorescence microscopy were performed to visualize cell viability. The scale bars represent 100 μm. Magnification = 20×. B. CCK-8 cell viability data for the same treatment groups. Error bars represent the standard deviation over three independent experiments with five replicates per experiment (n = 3). *** indicates p < 0.001 among indicated treatment groups. Group comparisons are indicated as determined by a one-way analysis of variance (ANOVA) with a Student−Newman−Keuls comparisons post hoc test.

Figure 7.

A. Bright field, fluorescence, and merged images showing fluorescence in C6 rat glioma cells preincubated with either PBS (blank), AAN-PTC–Fe²⁺ (with cleavable site and H₂S donor, 200 μM), AAQ-PTC–Fe²⁺ (with H₂S donor but no cleavable site, 200 μM), AAN-PC–Fe²⁺ (with cleavable site but no H₂S donor, 200 μM), FeCl₂ (200 μM), and AAN-PTC (with cleavable site and H₂S donor but no Fe²⁺, 200 μM) for 3 h. After treatment, cells were incubated with ROS probe DHE (10 μM) for 30 min, washed, and imaged in DPBS by bright-field and fluorescence microscopy. Scale bar is 100 μm. B. Corrected total cell fluorescence (CTCF) quantification for the same treatment groups. Average fluorescence intensities were quantified using ImageJ (cell counts are >30 for each group from three separate wells). The error bars represent the standard deviation among repeats (n=3). *** indicates p < 0.001 for a comparison of AAN-PTC–Fe²⁺ versus all other groups, which was determined by a one-way analysis of variance (ANOVA) with a Student–Newman–Keuls comparisons post hoc test.

Scheme 1.

Synthetic route to AAN-PTC and COS/H₂S release triggered by legumain and aided by CA. A detailed synthetic route to AAN-PTC appears in the supporting information.