pH and ROS sequentially responsive podophyllotoxin prodrug micelles with surface charge-switchable and self-amplification drug release for combating multidrug resistance cancer


Abstract

Multidrug resistance (MDR) is one of the main reasons for tumor chemotherapy failure. Podophyllotoxin (PPT) has been reported that can suppress MDR cancer cell growth; however, effective delivery of PPT to MDR cancer cells is challenged by cascaded bio-barriers. To effectively deliver PPT to MDR cancer cells, a PPT polymeric prodrug micelle (PCDMA) with the charge-conversion capability and self-acceleration drug release function are fabricated, which is composed of a pH and reactive oxygen species (ROS) sequentially responsive PPT-polymeric prodrug and an ROS generation agent, cucurbitacin B (CuB). After reach to tumor tissue, the surface charge of PCDMA could rapidly reverse to positive in the tumor extracellular environment to promote cellular uptake. Subsequently, the PCDMA could be degraded to release PPT and CuB in response to an intracellular high ROS condition. The released CuB is competent for generating ROS, which in turn accelerates the release of PPT and CuB. Eventually, the released PPT could kill MDR cancer cells. The in vitro and in vivo studies demonstrated that PCDMA was effectively internalized by cancer cells and produces massive ROS intracellular, rapid release drug, and effectively overcame MDR compared with the control cells, due to the tumor-specific weakly acidic and ROS-rich environment. Our results suggest that the pH/ROS dual-responsive PCDMA micelles with surface charge-reversal and self-amplifying ROS-response drug release provide an excellent platform for potential MDR cancer treatment.

Keywords: Multidrug resistance; ROS-sensitive; charge reverse; pH-responsive; polymeric prodrug.

Conflict of interest statement

The authors report no conflict of interest.

Figures

Scheme 1.
Scheme 1.
Illustration of the charge-conversion PCDMA system with self-amplifiable drug release for overcoming MDR in?vivo.
Figure 1.
Figure 1.
Characterization of micelles. The size distribution of PCDMA (A), PDMA (B), and PCSA(C) in PBS at pH 7.4. (D) Size changes of PCDMA after incubation with or without 10?mM H2O2 for different time. PPT (E) and CuB (F) release from PCDMA under different ROS conditions or pH conditions. Data are shown as mean?±?SD, n=?3.
Figure 2.
Figure 2.
Charge-switchable PCDMA micelles. Surface zeta potential changes of PCDMA (A) and PCSA (B) at pH 7.4 or 6.8 at different incubation times. (C) The amount of BSA adsorbed on the PCDMA and PCSA micelles after incubation at pH 7.4 or 6.8 (n=?6, ***p<?.001).
Figure 3.
Figure 3.
(A) CLSM images of A549/PTX cells after treatment with PCDMA and PCSA at pH 7.4 or 6.8. FCM results of A549/PTX cells after treatment with PCDMA and PCSA at pH 6.8 (B) or 7.4 (C). n=?6, ***p<?.001.
Figure 4.
Figure 4.
Evaluation of the ROS regenerating ability of CuB in?vitro and intracellular self-amplification drug release of PCDMA. FCM results of A549/PTX cells treated with CuB for different concentration for 2?h (A) or 0.1?μg/mL of CuB for different incubation time (B), n=?6. CLSM images (C) and FCM analysis (D) of A549/PTX cells stained with DCFH-DA after treatment with blank culture medium, CuB, PCDMA, PDMA, or PCSA for 4?h (n=?6, ***p<?.001). (E) PPT release amount of PCDMA and PDMA in A549/PTX cells after 8, 12, and 24?h incubation (n=?3, **p< .01; ***p<?.001).
Figure 5.
Figure 5.
In vitro cytotoxicity assay. Cell viability of A549/PTX and A549cells after treatment with PTX (A), PPT (B), or PCDMA (C) at pH 7.4 for 48?h. Cell viability of A549/PTX following treatment with PCDMA and PCSA at pH 7.4 (D) and 6.8 (E) for 4?h. (F) Cell viability of A549/PTX following treatment with PCDMA and PDMA at pH 7.4 for 48?h. n=?6, ***p<?.001.
Figure 6.
Figure 6.
MTD study. The survival rate of Kunming mice after treated with free PPT (A), PCDMA (B), PDMA (C), and PCSA (D). The body changes of Kunming mice after treated with free PPT (E), PCDMA (F), PDMA (G), and PCSA (H). n=?10.
Figure 7.
Figure 7.
In vivo antitumor efficiency. Tumor volume changes (A), tumor weight at day 14 (B), TSR (C), and body weight changes (D) during the study. n=?6, **p<?.01, ***p<?.001.
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