NIR-guided dendritic nanoplatform for improving antitumor efficacy by combining chemo-phototherapy

Abstract

Background: Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), is a promising noninvasive strategy in the treatment of cancers due to its highly localized specificity to tumors and minimal side effects to normal tissues. However, single phototherapy often causes tumor recurrence which hinders its clinical applications. Therefore, developing a NIR-guided dendritic nanoplatform for improving the phototherapy effect and reducing the recurrence of tumors by synergistic chemotherapy and phototherapy is essential.

Methods: A fluorescent targeting ligand, insisting of ICG derivative cypate and a tumor penetration peptide iRGD (CRGDKGPDC), was covalently combined with PAMAM dendrimer to prepare a single agent-based dendritic theranostic nanoplatform iRGD-cypate-PAMAM-DTX (RCPD).

Results: Compared with free cypate, the resulted RCPD could generate enhanced singlet oxygen species while maintaining its fluorescence intensity and heat generation ability when subjected to NIR irradiation. Furthermore, our in vitro and in vivo therapeutic studies demonstrated that compared with phototherapy or chemotherapy alone, the combinatorial chemo-photo treatment of RCPD with the local exposure of NIR light can significantly improve anti-tumor efficiency and reduce the risk of recurrence of tumors.

Conclusion: The multifunctional theranostic platform (RCPD) could be used as a promising method for NIR fluorescence image-guided combinatorial treatment of tumor cancers.

Keywords: ICG derivatives; chemotherapy; combined therapy; dendrimer; phototherapy.

Figure 1

(A) Schematic representation of the synthesis of theranostic platform RCPD for combined chemo-photo treatment of cancer cells. (B) UV−Vis absorption and fluorescence spectra of RCPD. (C) Size distribution of RCPD. (D) Drug release profiles of DTX from DTX suspension, RCPD (pH=5.5 or 7.4), PAMAM@DTX.

Figure 2

(A) Fluorescence intensity ratio of cypate and RCPD with PBS (0.16 mM cypate-equiv.) before (dark) and after (light) NIR irradiation (1.6 W/cm², 5 mins). **p<0.01, ***p<0.001. (B) Singlet oxygen generation by RCPD (0.02–0.16 mM cypate-equiv.) after NIR irradiation (1.6 W/cm², 5 mins). (C) Singlet oxygen generation by RCPD (0.16 mM cypate-equiv.) after NIR irradiation with different power density (0.3, 0.96, 1.6 W/cm², 5 mins). (D) Temperature change curves of PBS, cypate, and RCPD aqueous solution (0.16 mM cypate-equiv.) exposed to laser at a power density of 1.6 W/cm². (E) Temperature change curves of RCPD aqueous solution (0.02–0.16 mM cypate-equiv.) exposed to laser at a power density of 1.6 W/cm². (F) Temperature change curves of RCPD aqueous solution (0.16 mM cypate-equiv.) exposed to laser at the following power densities: 0.3, 0.96 and 1.6 W/cm².

Figure 3

(A) Confocal images of HepG2 cells after 1 hr, 2 hrs, 4 hrs, and 12 hrs of incubation with RCPD. Scale bar is 40 μm. (B) The confocal microscopy analysis of cellular uptakes of RCPD at different time.

Figure 4

(A) Confocal images of ROS generation in HepG2 cells at 24 hrs after various treatments. Scale bar is 50 μm. (B) The flow cytometric analysis of intracellular ROS productions in HepG2 cells treated with RCPD or cypate after NIR irradiation (1.6 W/cm², 5 mins). (C) Temperature viabilities of HepG2 cells treated with PBS, RCPD, or cypate. Abbreviations: ROS, reactive oxygen species.

Figure 5

(A) The LIVE-DEAD analysis of HepG2 cells at 24 hrs after various treatments. Scale bar is 100 μm. (B) The cell viability of DTX-resistant human liver cancer cells at 24 hrs after various treatments (*p<0.05). (C) Stacked bars of HepG2 cells apoptosis determined by flow cytometry using Annexin V-FITC and PI. (D) The cell apoptosis quantified by the flow cytometry at 24 hrs after various treatments. The cypate and DTX concentrations were 32 μM and 18.6 μM, respectively. In all experiments, PDT was irradiated with 808 nm, 0.3 W/cm², PDT+PTT was irradiated with 808 nm, 1.6 W/cm². Abbreviations: PTT, photothermal therapy; PDT, photodynamic therapy.

Figure 6

(A) The confocal images of SOSG-stained sections at 6 hrs after various treatments. Scale bars represent 100 μm. (B) Temperature change curves in tumors exposed to the 808 nm laser at a power density of 0.3 W/cm² and 1.6 W/cm² after intravenous injections of saline, RCP, and RCPD. Abbreviations: SOSG, singlet oxygen sensor green.

Figure 7

(A) The tumor growth curves of mice after various treatments. (B) The body weight changes of mice during various treatments. (C) The photos of mice and excised tumors after various treatments. The red circles indicate the tumor size. (D) The images of H&E stained tumor sections after various treatments. Scale bars represent 50 μm. The doses of cypate and DTX in all the above experiments were 6.7 and 5 mg/kg. The laser irradiation was carried out on the tumor at 808 nm at a power density of 0.3 W/cm² or 1.6 W/cm² for 5 mins. -*p<0.05, **p<0.01, ***p<0.001 for comparison to the control-.