Self-Luminescing Theranostic Nanoreactors with Intraparticle Relayed Energy Transfer for Tumor Microenvironment Activated Imaging and Photodynamic Therapy

Abstract

The low tissue penetration depth of external excitation light severely hinders the sensitivity of fluorescence imaging (FL) and the efficacy of photodynamic therapy (PDT) in vivo; thus, rational theranostic platforms that overcome the light penetration depth limit are urgently needed. To overcome this crucial problem, we designed a self-luminescing nanosystem (denoted POCL) with near-infrared (NIR) light emission and singlet oxygen (¹O₂) generation abilities utilizing an intraparticle relayed resonance energy transfer strategy. Methods: Bis[3,4,6-trichloro-2-(pentyloxycarbonyl) phenyl] oxalate (CPPO) as a chemical energy source with high reactivity toward H₂O₂, poly[(9,9'-dioctyl-2,7-divinylene-fluorenylene)-alt-2-methoxy- 5-(2-ethyl-hexyloxy)-1,4-phenylene] (PFPV) as a highly efficient chemiluminescence converter, and tetraphenylporphyrin (TPP) as a photosensitizer with NIR emission and ¹O₂ generation abilities were coencapsulated by self-assembly with poly(ethyleneglycol)-co-poly(caprolactone) (PEG-PCL) and folate-PEG-cholesterol to form the POCL nanoreactor, with folate as the targeting group. A series of in vitro and in vivo analyses, including physical and chemical characterizations, tumor targeting ability, tumor microenvironment activated imaging and photodynamic therapy, as well as biosafety, were systematically investigated to characterize the POCL. Results: The POCL displayed excellent NIR luminescence and ¹O₂ generation abilities in response to H₂O₂. Therefore, it could serve as a specific H₂O₂ probe to identify tumors through chemiluminescence imaging and as a chemiluminescence-driven PDT agent for inducing tumor cell apoptosis to inhibit tumor growth due to the abnormal overproduction of H₂O₂ in the tumor microenvironment. Moreover, the folate ligand on the POCL surface can further improve the accumulation at the tumor site via a receptor-mediated mechanism, thus enhancing tumor imaging and the therapeutic effects both in vitro and in vivo but without any observable systemic toxicity. Conclusion: The nanosystem reported here might serve as a targeted, smart, precise, and noninvasive strategy triggered by the tumor microenvironment rather than by an outside light source for cancer NIR imaging and PDT treatment without limitations on penetration depth.

Keywords: H2O2; PDT; chemiluminescent imaging; intraparticle relayed energy transfer; self-luminescing theranostic nanoreactors.

Scheme 1

(A) Schematic representation of the nanoreactor formulation (POCL), chemical structures of the constituents, and the principle of chemiluminescence and ¹O₂ generation of POCL in the presence of H₂O₂. (B) Schematic view of POCL luminescence and application in photodynamic therapy (PDT) specifically in tumors triggered by H₂O₂ after targeted delivery through intravenous injection. (C) Mechanism of the NIR chemiluminescence and PDT.

Figure 1

(A) The energy levels of PFPV, TPP and the high-energy intermediate DOTD. (B) Normalized emission and absorption spectra of PFPV and TPP, respectively. (C) Fluorescence spectra of POCL with different TPP dopant ratios vs. PFPV. (D) TEM image of POCL. (E) Size distribution of POCL in water measured by DLS. (F) Average hydrodynamic diameters of POCL as a function of time after storage in PBS with 10% FBS. (G) UV-vis absorption spectra of POCL, free PFPV, free TPP, and free CPPO. (H) CL images of POCL upon the existence of various concentrations of H₂O₂ collected by the ChemiDocTM MP Imaging System (BIO-RAD) with a 10 min exposure by open filter (top inset), and a correlation between the amounts of H₂O₂ and the corresponding gray values was calculated by ImageJ software (bottom inset). (I) Chemiluminescence spectra of POCL with different TPP dopant ratios vs. PFPV by the addition of an excess of H₂O₂ (1 M). The top inset is a photograph of the generated CL taken by a camera.

Figure 2

(A) UV-Vis spectra of ABDA and (B) normalized absorbance of ABDA at 380 nm in POCL solutions after the addition of H₂O₂ at different concentrations (*P < 0.05, **P < 0.01, ***P < 0.001; n = 3 per group). (C) UV-Vis spectra of ABDA and (D) normalized absorbance of ABDA at 380 nm in POCL, POCL/CPPO-, POCL/PFPV- or POCL/TPP- solutions after the addition of 8 μM H₂O₂ (*P < 0.05, **P < 0.01, ***P < 0.001; n = 3 per group). (E) CLSM images of HeLa cells after incubation with POCL or POCL/FA- for 4 h. Scale bar = 20 µm.

Figure 3

(A) The cytotoxicity of POCL against LO2 cells after incubation for 24 h or 48 h. (B) The cytotoxicity of POCL against HeLa cells at various PFPV concentrations in the presence or absence of 0.2 μM H₂O₂ (*P < 0.05, **P < 0.01, ***P < 0.001; n = 4 per group). (C) The cytotoxicity of different kinds of nanoparticles (expressed as POCL/CPPO-, POCL/PFPV-, POCL/TPP-, POCL, or POCL/FA-) against HeLa cells in the presence of 0.2 μM H₂O₂. All concentrations of nanoparticles were kept the same with the counterpart PFPV of 40 μg/mL (*P < 0.05, **P < 0.01, ***P < 0.001; n = 4 per group). (D) Fluorescence microscopic images of HeLa cells stained by the live/dead cell staining kit after different treatments as mentioned above. Scale bar = 100 µm. (E) Evaluation of the cell death mechanisms through Annexin V-FITC and PI staining after different treatments as mentioned above. (F) Intracellular ROS generation mediated by different treatments as mentioned above. Scale bar = 100 µm.

Figure 4

(A) Time course fluorescence images of tumor-bearing mice receiving i.v. injection of POCL or POCL/FA- (0.2 mg/mL based on TPP, 100 μL per mouse). Tumor regions are indicated by white arrows. Fluorescence images were acquired for 1 s of exposure with excitation at 465 nm and emission at 640 nm. (B) Fluorescence signal intensities of the tumors in mice from A over time (*P < 0.05, **P < 0.01, ***P < 0.001; n = 3 per group). (C) Ex vivo fluorescence images and (D) corresponding fluorescence signal intensities of the dissected organs and tumors from the mice after 24 h of injection as indicated above (*P < 0.05, **P < 0.01, ***P < 0.001; n = 3 per group). (E) Biodistribution of POCL or POCL/FA- based on TPP at 24 h post-injection as indicated above (n = 3 per group). (F) Pharmacokinetics of POCL or POCL/FA- based on TPP in mice from 0 to 12 h after intravenous injection (n = 3 per group). (G) Time course chemiluminescence images of tumor-bearing mice receiving i.v. injection of POCL or POCL/FA- (0.2 mg/mL based on TPP, 100 μL per mouse). Tumor regions are indicated by white arrows. (H) Chemiluminescence signal intensities of the tumor in mice from G over time (*P < 0.05, **P < 0.01, ***P < 0.001; n = 3 per group).

Figure 5

In vivo tumor therapeutic effect. (A) Schematic illustration of the treatment regimen. The i.v. injection was implemented on an every-three-day schedule four times (TPP dose: 4 mg/kg). The mice were sacrificed on day 21 for tumor separation. (B) Tumor growth curves with different treatments (*P < 0.05, **P < 0.01, ***P < 0.001; n = 5 per group). (C) Representative tumor images. (D) Tumor weight after 21 d of different treatments (*P < 0.05, **P < 0.01, ***P < 0.001; n = 5 per group). (E) Optical microscopic images of tumor slices stained by H&E, Ki67 antigen immunohistochemistry, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling (TUNEL). Scale bar = 50 μm.