Molecular domino reactor built by automated modular synthesis for cancer treatment

Abstract

Rationale: A cascade, or domino, reaction consists of two, or more, consecutive reactions such that subsequent reactions occur only if some chemical functionality has first been established in the prior step. However, while construction of predesigned and desired molecular domino reactors in a tailored manner is a valuable endeavor, it is still challenging. Methods: To address this challenge, we herein report an aptamer-based photodynamic domino reactor built through automated modular synthesis. The engineering of this reactor takes advantage of the well-established solid-phase synthesis platform to incorporate a photosensitizer into G-quadruplex/ hemin DNAzyme at the molecular level. Results: As a proof of concept, our photodynamic domino reactor, termed AS1411/hemin- pyrochlorophyll A, achieves in vivo photodynamic domino reaction for efficient cancer treatment by using a high concentration of hydrogen peroxide (H₂O₂) in the tumor microenvironment (TME) to produce O₂, followed by consecutive generation of singlet oxygen (¹O₂) using the pre-produced O₂. More specifically, phosphoramidite PA (pyrochlorophyll A) is coupled to aptamer AS1411 to form AS1411-PA ApDC able to simultaneously perform in vivo targeted imaging and photodynamic therapy (PDT). The insertion of hemin into the AS1411 G-quadruplex was demonstrated to alleviate tumor hypoxia by decomposition of H₂O₂ to produce O₂. This was followed by the generation of ¹O₂ by PA to trigger cascading amplified PDT. Conclusion: Therefore, this study provides a general strategy for building an aptamer-based molecular domino reactor through automated modular synthesis. By proof of concept, we further demonstrate a novel method of achieving enhanced PDT, as well as alleviating TME hypoxia at the molecular level.

Keywords: Automated modular synthesis; Molecular domino reactor; Molecular “elements”; Photodynamic therapy; Tumor microenvironment (TME).

Figure 1

(A) Scheme illustrating automated and modular synthesis of AS1411/hemin-PA ApDC as a molecular domino reactor and its application for cascading amplified photodynamic therapy. Inside the tumor microenvironment, AS1411/hemin-PA, as a photodynamic domino reactor, can catalyze two consecutive reactions, i.e., decomposition of inherent H₂O₂ to produce O₂ and utilization of produced O₂ to generate ¹O₂ for cascading amplified PDT. (B) Synthetic route of phosphoramidite PA as a module for DNA solid-phase synthesis. Pyrochlorophyll-A (PA), a photosensitizer, was linked with solid-phase synthesis functionalities to form modular phosphoramidite-PA for automated solid-phase synthesis of AS1411-PA ApDC. Subsequently, hemin, an iron-containing porphyrin, was inserted into the G-rich region of AS1411 to form AS1411/hemin-PA with G-quadruplex/hemin DNAzyme structure.

Figure 2

Characterization of AS1411/hemin-PA. (A) HPLC chromatogram of AS1411-PA conjugate. (B) UV-vis-NIR spectra of hemin, AS1411-PA, and AS1411/hemin-PA. (C) Oxygen generation in hydrogen peroxide solution with or without addition of AS1411/hemin-PA at room temperature. (D) Singlet oxygen generation, as determined by the increased SOSG fluorescence of AS1411-PA and AS1411/hemin-PA with or without hydrogen peroxide under 670-nm NIR light irradiation.

Figure 3

(A) Scheme of aptamer-based molecular domino reactor to generate ¹O₂. (B) Flow cytometry assay showing the binding affinities of MCF-7 cell after incubation of AS1411-0T-PA, AS1411-4T-PA, AS1411-10T-PA, AS1411-20T-PA, AS1411-40T-PA. (C) Singlet oxygen generation, as determined by the increased SOSG fluorescence, of AS1411/hemin-4T-PA, AS1411/hemin-10T-PA, AS1411/hemin-20T-PA, and AS1411/hemin-40T-PA in the presence of hydrogen peroxide under 670-nm NIR light irradiation for 10 min. P values in (C) were calculated by Tukey's post-test (***P < 0.001, **P < 0.01 or *P < 0.05).

Figure 4

AS1411/hemin-PA for in vitro enhanced PDT. (A) Scheme illustrating targeted delivery of PA and cascading PDT. (B) Confocal images of MCF-7 cells incubated with free PA, AS1411-PA, or AS1411/hemin-PA with the same concentration of PA. Blue and red colors represent DAPI-stained cell nuclei and PA fluorescence, respectively. (C) Flow cytometric analysis of MCF-7 cells and 293T cells after treatment with 400 nM free PA, AS1411-PA or AS1411/hemin-PA in cell growth media for 1h. (D) in vitro PDT treatment of MCF-7 cells after treatment with free PA, AS1411-PA or AS1411/hemin-PA in the presence or absence of 100 μM H₂O₂ under 670-nm NIR light irradiation for 30 min at the power density of 5 mW/cm². P values in (D) were calculated by Tukey's post-test (***P < 0.001, **P < 0.01 or *P < 0.05).

Figure 5

In vivo imaging and biodistribution of MCF-7-bearing mice after i.v. injection of AS1411/hemin-PA. (A) In vivo fluorescence imaging of mice bearing MCF-7 tumor at various time points post-injection of PA or AS1411/hemin-PA. (B) Relative fluorescence intensities of the tumors from different groups of mice at different time points based on in vivo fluorescence images shown in (A). (C) Ex vivo imaging of major organs and tumor extracted at 5 h post-injection of PA or AS1411/hemin-PA. (D) The biodistribution profiles of AS1411/hemin-PA in MCF-7-bearing mice at 5 h p.i. H, Li, Sp, Lu, Ki, St, I, M, Sk, B, and T stand for heart, liver, spleen, lung, kidneys, stomach, intestine, muscle, skin, bone, and tumor, respectively.

Figure 6

In vivo enhanced photodynamic therapy with AS1411/hemin-PA. (A) Representative immunofluorescence images of tumor slices extracted from mice treated with AS1411-PA or AS1411/hemin-PA after hypoxia staining. Blood vessels and hypoxic areas were stained by antiCD31 antibody (red) and anti-pimonidazole antibody (green), respectively. (B) Quantification of hypoxic areas in each group was recorded from more than 10 images using ImageJ software. (C) The tumor growth curves for various groups of mice (five mice per group) are indicated. (D) Average weights of mouse tumor in different groups at 16 days after various treatments. (E) Micrographs of H&E- and TUNEL-stained tumor slices extracted from various groups on the second day post-treatment. P values in (B,C,D) were calculated by Tukey's post-test (***P < 0.001, **P < 0.01 or *P < 0.05).