pH/Redox Dual-Responsive Drug Delivery System with on-Demand RGD Exposure for Photochemotherapy of Tumors

Abstract

Purpose: Nanotechnology has been widely used in antitumor research. The complex physiological environment has brought significant challenges to the field of antitumor micelles. The ideal micelles must not only have an invisible surface to extend the circulation time but must also enhance the retention of drugs and cellular internalization at the tumor.

Methods: A graded response micelle (RPPssD@IR780/DOC) was designed to self-assemble by cRGD-poly(β-amino esters)-polyethylene glycol-ss-distearoyl phosphatidylethanolamine (cRGD-PBAE-PEG-ss-DSPE) loaded with docetaxel (DOC) and IR-780 iodide (IR780). The micelles were designed to allow the PEG shell to prolong the blood circulation time in the body and effectively accumulate in the tumor. Subsequently, the acidic microenvironment of the tumor could transform the PBAE to hydrophilic, thereby increasing the size of micelles and exposing cyclic Arg-Gly-Asp (cRGD) peptides to increase the retention and cellular internalization of micelles in the tumor. After tumor cells had captured micelles, the high expression of glutathione in the cells prompted the release of DOC and IR780. Subsequently, the IR780 was stimulated by an 808-nm laser to generate local heat and reactive oxygen species (ROS) to synergize with DOC to treat the tumor.

Results: In vitro and in vivo experimental results suggested that RPPssD@IR780/DOC was a potential photochemical effective for the treatment of tumors with non-negligible antitumor activity and good biocompatibility.

Conclusion: A dual-response pH/redox delivery system with on-demand RGD exposure was designed to achieve photochemotherapy of tumors with good biosafety and antitumor effects.

Keywords: drug delivery; ligand hidden; micelles; photo-chemotherapy; size variable.

Graphical abstract

Figure 1

Schematic illustration of RPPssD@IR780/DOC micelles to achieve particle size changes, hide targets, and treat tumor synergistic photochemical combination. (A) Formulation, particle size changes, hidden targets, and degradation mechanism of RPPssD@IR780/DOC micelles. (B) The tumor-targeted micelles RPPssD loaded with DOC and IR780 to treat tumors through light-triggered photochemotherapy.

Figure 2

Characterization of RPPssD NPs. (A) The CMC of cRGD-PBAE-PEG-ss-DSPE. (B) The hydrodynamic diameter of RPPssD@IR780/DOC and the blank micelles of RPPssD. (C) TEM image of RPPssD@IR780/DOC (Scale bar=200 nm). (D) Zeta potential of RPPssD@IR780/DOC and blank micelles of RPPssD. (E) The size and PDI of RPPssD@IR780/DOC in PBS for 30 days. (F) The size and PDI of RPPssD@IR780/DOC medium containing 10% fetal bovine serum for 7 days. Data are presented as mean ± standard deviation (n = 3).

Figure 3

Performance evaluation of RPPssD NPs. (A) Variation curves of RPPssD particle size at different pH values and TEM of RPPssD at pH 4.5 (Scale bar=500 nm) and 6.5 (Scale bar=200 nm). (B) Acid-base titration curve of RPPssD. (C) The curve of RPPssD NP size changes with time at different concentrations of DTT. (D) Infrared thermogram of each sample solution after NIR irradiation. (E) Temperature change curves of each sample solution under NIR irradiation. (F) Temperature change curves of free IR780 and RPPssD@IR780 after 3 cycles of laser irradiation. (G) Temperature rise and fall curves of IR780 and RPPssD@IR780/DOC aqueous dispersion. (H) The linear time data versus –lnθ obtained from the cooling period of free IR780. (I) The linear time data versus –lnθ obtained from the cooling period of RPPssD@IR780/DOC. (J) The remaining amount of DPBF after 10 minutes of laser irradiation for different samples. (K) DOC-release curves from RPPssD@IR780/DOC at varying conditions. (L) Release curves of the IR780 from RPPssD@IR780/DOC at varying conditions. (M) The hemolysis rate and experimental hemolysis phenomenon of RPPssD. (N) Cell viability of MDA-MB-231 cells after treatment with RPPssD. (O) Cell viability of L02 cells after treatment with RPPssD. Data are presented as mean ± standard deviation (n = 3), ns > 0.05, *p < 0.05.

Figure 4

In vitro cellular experiments. (A) Uptake of RPPssD@IR780 and IR780 at different pH levels by MDA-MB-231 cells and MCF-7 cells. (B) Cell uptake of MDA-MB-231 cells was quantified by Image J. (C) The mean fluorescence intensity of the formulation uptake by MCF-7 cells was determined by Image J. (D) The ROS in MDA-MB-231 cells after treatment with different samples were detected by DCFH-DA. (E) The cytotoxicity of RPPssD@IR780/DOC and free DOC/IR780 for MDA-MB-231 cells. (F) The cytotoxicity of RPPssD@IR780/DOC and free DOC/IR780 for MCF-7 cells. (G) Fluorescence images of MDA-MB-231 cells after treatment with the formulations (The concentration of DOC and IR780 was 1 μg/mL and 0.2 μg/mL, respectively.) and Calcein/PI staining. (Scale bar = 200 μm). Data are presented as mean ± standard deviation (n = 3), ns > 0.05, *p < 0.05, **p < 0.01 ***p < 0.001.

Figure 5

Evaluation of in vivo thermal activity and antitumor effect. (A) Infrared thermograms of tumor-bearing mice after infrared laser irradiation with saline injection into the tail vein, free DOC/IR780, and RPPssD@IR780/DOC, respectively. (B) Change curves of tumor surface temperature in different groups of tumor-bearing mice after infrared laser irradiation. (C) Experimental protocol for in vivo photochemical therapy of tumors. (D) Tumor volume variation curves of tumor-bearing mice in each group. (E) The body weight variation of tumor-bearing mice in each group. (F) Tumor weight of tumor-bearing mice in each group. (G) Survival curves of tumor-bearing mice in each group. (H) Photographs of tumor tissues after treatment in different groups. (I) The H&E staining and Ki67 immunohistochemical analysis of tumor tissues from tumor-bearing mice in each treatment group (Scale bar=50 μm). Data are presented as mean ± standard deviation (n = 6), ns > 0.05, ***p < 0.001.

Figure 6

Biosafety of RPPssD@IR780/DOC micelles. (A) H&E staining of major organ tissues (heart, liver, spleen, lung, and kidney) of tumor-bearing mice in each treatment group. (Scale bar=50 μm). (B–L) Routine and blood biochemical parameters of mice after tail vein injection of saline, free DOC/IR780 and RPPssD@IR780/DOC with or without laser irradiation. ALT, AST, and ALP were used to assess liver injury, renal injury was estimated by BUN and CRE, while CK was an indicator of myocardial injury. In addition, WBC, RBC, HGB, NEU% and PLT were used in blood routine to evaluate the toxicity of the drug in mice. Data are presented as mean ± standard deviation (n = 6).