ROS-triggered self-accelerating drug release nanosystem with charge conversion for enhanced cancer therapy

Background: The enhancement tumor retention and of cellular uptake of drugs are important factors in maximizing anticancer therapy and minimizing side effects of encapsulated drugs. Herein, a delivery nanoplatform with a pH-triggered charge-reversal capability and self-amplifiable reactive oxygen species (ROS) level inducing drug release pattern was constructed by encapsulating doxorubicin (DOX) in pH/ROS-responsive polymeric micelle. Results: The surface charge of this system can be converted from negative to positive for enhanced tumor cell uptake in response to the weakly acidic tumor tissue. In addition, methionine-based system was dissociated in a ROS-rich intracellular environment, resulting in a phase transition and the release of DOX. Then, the exposed α -tocopheryl succinate ( α -TOS) segments can be capable of producing ROS, which further induced the self-amplifiable disassembly of the micelles and drug release. Conclusions: We confirmed efficient DOX delivery into cancer cells, upregulation of tumoral ROS level and induction of the apoptotic capability in vitro . The system exhibited outstanding tumor inhibition capability in vivo , indicating that dual stimuli nanosytem would be great potential as an anticancer drug delivery platform.


Micelle preparation and characterization
The solvent exchange method was used to prepare micelles in this study.
DOX·HCl ( The drug-loading content (DLC) and drug-loading efficiency (DLE) of DOX micelles were calculated by the following equations.
( For the characterizations of the empty micelle or DOX-loaded micelles, the particle sizes, size distributions and zeta-potentials were measured by using a Zetasizer (Malvern 3000HSA). The morphologies of the micelles were identified by transmission electron microscopy (TEM) images obtained using a JEM-2000EXII transmission electron microscope with an accelerating voltage of 200 kV. The morphology changes of micelles were also evaluated when micelles were incubated in different pH solutions or H2O2 solutions.

pH-sensitive property of PPT/D(DMA)@DOX
In order to evaluate pH-sensitive property of PPT/D(DMA)@DOX, the micelle was tested compared with PPT/D(SA)@DOX. Two above micelles (500 μg/mL) were incubated in PBS at pH 7.4 and 6.8 for 200 min, respectively. At predetermined intervals, the mean diameter and zeta potentials of the micelles were measured by DLS.

Drug release behavior
To study the ROS responsibility of PPT/D(DMA)@DOX, 3.0 mg of micelles

Cell culture
Human lung adenoma cell lines A549 were purchased from the American Type Culture Collection (ATCC, Rockville). The cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin and grown in a 37°C humidified environment containing 5% CO2. The cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. All the cytotoxicity tests were conducted in triplicate.

In vivo antitumor study and histochemistry analysis
Nude mice (5-6 weeks old) were purchased from Beijing Institution for Drug

Statistical analysis
The results were presented as mean ± standard deviation (SD). Statistical significance was analyzed using Student's t-test.

Synthesis and characterization of micelles
In this study, we have synthesized a biocompatible pH/ROS-responsive micelle via ROP polymerization and stepwise chemical grafting reactions as illustrated in  Table 1 and Figure 1C, D. The mean diameter of empty micelle was found to be 98.1 ± 4.5 nm. When DOX was encapsulated in the nanocarriers in proportion of 10%, the mean size of micelles was decreased to 84.3 ± 3.6 nm with a particle size distribution of PDI = 0.108. The DLC and DLE were calculated to be 9.64% and 96.4%, respectively.
To investigate the sensitivity of micelles to pH and ROS, TEM and DLS were used to study the morphology and size change of PPT/D(DMA)@DOX micelle in response to pH and ROS stimuli. The micelle was characterized in terms of size and surface morphology at pH 6.8 after 24 h incubation and with H2O2 for 6 h as shown in Figure 1E, F. At pH 7.4, the average size of the PPT/D(DMA)@DOX was 84.3 ± 3.6 nm, which increased to 116.2 ± 6.3 nm at pH 6.8. This might result from the swell of exposed amine from methionine segments with positive charge after the cleavage of DMA groups from the micelle at pH 6.8 [27] . Similarly, the TEM image also revealed increase in the size at pH 6.8. Moreover, PPT/D(DMA)@DOX exhibited a disintegrated morphology in the existence of H2O2. The destruction of the micelle structures was also determined with DLS and two peaks containing a large aggregated form were observed, indicating the high sensitivity of the micellar system to ROS.
The underlying mechanism was that thioether groups in methionine segments could be changed to hydrophilic sulfoxide groups by ROS and then induced the disassembly of micelle [20,28] . which could reduce the interaction with negatively charged proteins in the blood.

Apoptosis assay
In vitro antitumor activities of the PPT/D(DMA)@DOX micelles were analyzed by apoptosis assay using FITC Annexin V and Propidium Iodide (PI). As shown in PPT/D(DMA)@DOX micelles had relative high percentages of apoptotic cells, confirming the enhanced antitumor efficacy of PPT/D(DMA)@DOX system [34] .

In vitro cytotoxicity
To investigate the cytotoxicity of free DOX, PPT/D(SA)@DOX and PPT/D(DMA)@DOX, the MTT assay in A549 cells was performed at pH 7.4 and 6.8.
From Figure 5E, all the groups presented a DOX concentration-dependent growth inhibition effect on A549 cells. When the DOX concentration increased to 2 μg/mL, all micelles groups exhibited a lower cell viability than the free DOX group, indicating that the micelles improved the cytotoxicity of DOX. As for the free DOX h was 1.08 μg/mL and 0.75 μg/mL respectively at pH 6.8 meaning that the pH-sensitive micelles with charge reversal promoted the phagocytosis effect of tumor cells and enhanced the DOX accumulation in tumor cells [35] . had the strongest cellular uptake effect at pH 6.8, which was in accordance with the MTT results. These data demonstrated that the charge-reversal could obviously enhance cellular uptake in the tumor microenvironment [36] .  [37] .  [38] . Secondly, tumor acidity-activating charge conversion could effectively improve cell uptake of PPT/D(DMA)@DOX. Moreover, after internalization, the endogenous ROS would induce micelle disassembly and drug release, and the exposed α-TOS segments could further produce ROS for amplifying micelle disassembly and drug release [39,40] . Above reasons contributed to the superior therapeutic efficacy of PPT/D(DMA)@DOX system.

Conclusions
In summary, we developed a pH/ROS-responsive micelle drug delivery system with charge reversal and self-amplifiable drug release for tumor therapy. The micelle with negatively charged surface in blood had a great ability of prolonging circulation time; the charge reversal occurred followed by the exposure of positively charged amine of methionine due to pH responsiveness in tumor tissue, resulting in excellent cell membrane penetrating. It was found that owing to the ROS-responsive thioether groups, this designed nano-system could disassemble and deliver the drug to tumor cells and produce the cell toxicity. Moreover, exposed α-TOS segments would lead to the augmented concentration of intracellular ROS and accelerate release of DOX. Due to its unique advantages such as efficient cellular uptake and triggering targeted drug release, this designed system with charge reversal will exhibit great potential for achieving better therapeutic effects in cancer treatment.