Single-Component Dual-Functional Autoboost Strategy by Dual Photodynamic and Cyclooxygenase-2 Inhibition for Lung Cancer and Spinal Metastasis

Abstract

Coloading adjuvant drugs or biomacromolecules with photosensitizers into nanoparticles to enhance the efficiency of photodynamic therapy (PDT) is a common strategy. However, it is difficult to load positively charged photosensitizers and negatively charged adjuvants into the same nanomaterial and further regulate drug release simultaneously. Herein, a single-component dual-functional prodrug strategy is reported for tumor treatment specifically activated by tumor microenvironment (TME)-generated HOCl. A representative prodrug (DHU-CBA2) is constructed using indomethacin grafted with methylene blue (MB). DHU-CBA2 exhibited high sensitivity toward HOCl and achieved simultaneous release of dual drugs in vitro and in vivo. DHU-CBA2 shows effective antitumor activity against lung cancer and spinal metastases via PDT and cyclooxygenase-2 (COX-2) inhibition. Mechanistically, PDT induces immunogenic cell death but stimulates the gene encoding COX-2. Downstream prostaglandins E₂ and Indoleamine 2,3 dioxygenase 1 (IDO1) mediate immune escape in the TME, which is rescued by the simultaneous release of indomethacin. DHU-CBA2 promotes infiltration and function of CD8⁺ T cells, thus inducing a robust antitumor immune response. This work provides an autoboost strategy for a single-component dual-functional prodrug activated by TME-specific HOCl, thereby achieving favorable tumor treatment via the synergistic therapy of PDT and a COX-2 inhibitor.

Keywords: COX‐2; HOCl response; immune activation; photodynamic therapy.

Scheme 1

Schematic of the preparation and use of DHU‐CBA2 for treating lung cancer and spinal metastasis. A) Synthesis routes and HOCl‐response release of DHU‐CBA2. B) Conceptual and effective mechanisms of DHU‐CBA2 in triggering immunogenic cell death and blocking immune escape to remodel the tumor microenvironment.

Figure 1

A) Schematic of the single‐component dual‐functional autoboost strategy. B) Fluorescence spectra of DHU‐CBA2 (5 µM) with different concentrations of HOCl. C) Absorption spectra of DHU‐CBA2 (5 µM in 10 mM PBS, pH 7.4) before and after the addition of 20 µM HOCl. D) Time‐dependent changes in fluorescent intensity of DHU‐CBA2 (5 µM) at 686 nm after adding HOCl (20 µM). E) Fluorescent intensity of DHU‐CBA2 (5 µM) at 686 nm after adding different ROS/RNS (40 µM) (from A to K: DHU‐CBA2 only, H₂O₂, TBHP, ROO•, NO, •OH, ONOO^–, TBO•, O^2–, 5 µM HOCl, and 20 µM HOCl). F) Fluorescent intensity of DHU‐CBA2 (5 µM) at 686 nm after adding different ions/amino acids (50 µM) (from A to N’: DHU‐CBA2 only, CH₃COO^–, CO₃ ^2–, SO₄ ^2–, F^–, Cl^–, I^–, NO₂ ^–, S₂O₃ ^2–, NH₄ ⁺, Na⁺, Mg²⁺, Al³⁺, K⁺, Ca²⁺, Fe³⁺, Cu²⁺, Ni²⁺, Leu, Pro, Gly, Gln, Glu, Met, Lys, Trp, Ser, Thr, Asp, Ile, Val, His, Ala, Cys, Phe, Asn, Tyr, Arg, 5 µM HOCl, and 20 µM HOCl). G) Fluorescence intensity of DHU‐CBA2 (5 µM) upon addition of four equiv. HOCl in the presence of 4 equiv. various amino acids (from A to U: DHU‐CBA2 only, Leu, Pro, Gly, Gln, Glu, Met, Lys, Trp, Ser, Thr, Asp, Ile, Val, His, Ala, Cys, Phe, Asn, Tyr, Arg). H) The absorbance of DHU‐CBA2 (5 µM) at 664 nm before/after addition of 20 µM HOCl in buffer with different pH. I) HPLC analysis of 5 µM MB, 5 µM free Ind, 5 µM DHU‐CBA2 only, and 5 µM DHU‐CBA2 + 20 µM HOCl in 10 mM PBS (at 254 nm).

Figure 2

HOCl‐response behavior of DHU‐CBA2 in vitro and in vivo. After incubation with DHU‐CBA2, the medium was replaced by PBS with HOCl for 15 min. A) The MB‐positive LLC cells were detected by flow cytometry after various treatments, and (B) were further quantitatively analyzed. Experimental data in B) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 replicates; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). C) The HOCl‐response behavior and (D) statistical analysis of DHU‐CBA2 were recorded by in vivo bioluminescence imaging in LLC‐bearing subcutaneous tumors. Experimental data in D) were presented as mean ± SD. Statistical significance was performed via one‐way ANOVA with Tukey's test. (n = 3 individual animals; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). E) The HOCl‐response behavior and F) statistical analysis of DHU‐CBA2 were recorded by in vivo bioluminescence imaging in LLC‐bearing subcutaneous tumors in spinal metastasis. Experimental data in (F) were presented as mean ± SD. Statistical significance was performed via one‐way ANOVA with Tukey's test. (n = 3 individual animals; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Figure 3

The cell killing and ICD induction capacity of activated DHU‐CBA2 with PDT in vitro. A) Schematic of the antitumor and ICD induction of DHU‐CBA2 with PDT in cancerous cells. The CCK8 results of the cell viability in LLC cells incubated with DHU‐CBA2 at different concentrations for 12 h B) and 24 h C). Experimental data in (B,C) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 5 replicates, ns represents no significant difference). D) Live/dead cell staining assay of LLC cells after treatment by various treatments (1: CTRL; 2: DHU‐CBA3+HOCl; 3: DHU‐CBA2+HOCl; 4: DHU‐CBA3+HOCl (+); 5: DHU‐CBA2+HOCl (+); “(+)” represents high‐dose laser irradiation). E) Quantitative determination of ATP secretion in the cell culture supernatant after various treatments. Experimental data were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 replicates; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). F,G) Immunofluorescence staining results of CRT exposure and HMGB1 release in LLC cells after various treatments.

Figure 4

Immune response mechanism of DHU‐CBA2 with PDT via alleviating the high concentration of PGE₂ and IDO1. A) Relative mRNA levels of Ptgs2, B) protein level of COX‐2, and C) the level of PGE₂ in the supernatant in LLC cells with MB (+). (+) represents low‐dose laser irradiation in vitro. Experimental data in (A and C) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 replicates, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). D) The protein level of PGE₂ in LLC‐bearing subcutaneous tumors by various treatments (1: CTRL; 2: DHU‐CBA3; 3: DHU‐CBA2; 4: DHU‐CBA3 (+); 5: DHU‐CBA2 (+); “(+)” represents laser irradiation). Experimental data were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). E) Association between CTL levels and overall survival of patients with lung cancer with different IDO1 gene copy numbers. Continuous z = 2.59; P = 0.00956. Z‐scores and p‐values were computed by the two‐sided Wald test in Cox‐PH regression. F) Representative images of IDO1 expression in subcutaneous tumors after various treatments by immunohistochemistry staining analysis. G) CD3⁺CD8⁺, and H) GranB⁺CD8⁺ T cells in subcutaneous tumors after various treatments were detected and quantified by flow cytometry. Experimental data in (G,H) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Figure 5

Therapeutic efficacy of DHU‐CBA2 in subcutaneous LLC‐bearing mice. A–E) Subcutaneous tumors by various treatments (1: CTRL; 2: DHU‐CBA3; 3: DHU‐CBA2; 4: DHU‐CBA3 (+); 5: DHU‐CBA2 (+); “(+)” represents laser irradiation): A) Schematic, B) bioluminescence images, and C) statistical analysis of tumorigenesis. Experimental data in C) were presented as mean ± SD. Statistical significance was calculated via two‐way ANOVA with Tukey's test. (n = 5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). (D) Tumor volume is recorded every two days. Experimental data were presented as mean ± SD. Statistical significance was calculated via two‐way ANOVA with Tukey's test. (n = 5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). (E) Tumor weight at the end of the observation period. Experimental data were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Figure 6

Therapeutic efficacy of DHU‐CBA2 in lung cancer spinal metastasis (LC‐SM). (A–H) LC‐SM tumors by various treatments (1: CTRL; 2: DHU‐CBA3; 3: DHU‐CBA2; 4: DHU‐CBA3 (+); 5: DHU‐CBA2 (+); “(+)” represents laser irradiation): A) Photographs of the tumors and B) tumor volume, at the end of the observation period. Experimental data in (B) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3–5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). C) Paralysis rate monitoring. Statistical significance was calculated via a log‐rank test for comparison. (n = 5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). D) 3D and planar view reconstruction images of spines showing the osteolytic vertebral plate (white arrow) and anterior centrum (red arrow) at the end of the observation period. (E) Bone mineral density (BMD) of lumbar three. Experimental data were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3–5 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). (F) TUNEL staining, (G) CD8⁺ T cells staining, and (H) IFN‐γ secretion. Experimental data in (H) were presented as mean ± SD. Statistical significance was calculated via one‐way ANOVA with Tukey's test. (n = 3 individual animals per group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).