Hypoxia-responsive lipid-poly-(hypoxic radiosensitized polyprodrug) nanoparticles for glioma chemo- and radiotherapy

Abstract

Treatment of malignant glioma is a challenge facing cancer therapy. In addition to surgery, and chemotherapy, radiotherapy (RT) is one of the most effective modalities of glioma treatment. However, there are two crucial challenges for RT facing malignant glioma therapy: first, gliomas are known to be resistant to radiation due to their intratumoral hypoxia; second, radiosensitizers may exhibit a lack of target specificity, which may cause a lower concentration of radiosensitizers in tumors and toxic side effects in normal tissues. Thus, novel angiopep-2-lipid-poly-(metronidazoles)n (ALP-(MIs)n) hypoxic radiosensitizer-polyprodrug nanoparticles (NPs) were designed to enhance the radiosensitizing effect on gliomas. Methods: In this study, different degrees and biodegradabilites of hypoxic radiosensitizer MIs-based polyprodrug (P-(MIs)n) were synthesized as a hydrophobic core. P-(MIs)n were mixed with DSPE-PEG2000, angiopep-2-DSPE-PEG2000 and lecithin to self-assemble ALP-(MIs)n through a single-step nanoprecipitation method. The ALP-(MIs)n encapsulate doxorubicin (DOX) (ALP-(MIs)n/DOX) and provoke the release of DOX under hypoxic conditions for glioma chemo- and radiotherapy. In vivo glioma targeting was tested in an orthotopic glioma using live animal fluorescence/bioluminescence imaging. The effect on sensitization to RT of ALP-(MIs)n and the combination of chemotherapy and RT of ALP-(MIs)n/DOX for glioma treatment were also investigated both in vitro and in vivo. Results: ALP-(MIs)n/DOX effectively accumulated in gliomas and could reach the hypoxic glioma site after systemic in vivo administration. These ALP-(MIs)n showed a significant radiosensitizing effect on gliomas and realized combination chemotherapy and RT for glioma treatment both in vitro and in vivo. Conclusions: In summary, we constructed a lipid-poly-(hypoxic radiosensitized polyprodrug) nanoparticles for enhancing the RT sensitivity of gliomas and achieving the combination of radiation and chemotherapy for gliomas.

Keywords: blood-brain barrier; chemo- and radiotherapy; glioma; hypoxia-responsive; radiosensitized polyprodrug.

Scheme 1

Schematic of the hypoxia-responsive and hypoxia RT sensitization ALP-(MIs)n drug-delivery system. (A) Mechanism of ALP-(MIs)n RT sensitization and DOX release under hypoxic condition and formation of ALP-(MIs)n/DOX. Six electrons are transferred in the complete reduction of nitro (R-NO₂) to amine (R-NH₂) under hypoxic conditions via a single-electron reduction catalyzed by a series of intracellular nitroreductases. (B) Formation of AL-PLGA/DOX as the control group. (C) Schematic illustrating ALP-(MIs)n applications: i) hypoxic cell radiosensitizer; ii) hypoxia-responsive release of DOX into the cytoplasm, and then transports it to the nucleus to kill tumor cells.

Figure 1

(A) Size distribution of AL-PLGA, ALP-(MIs)25, and ALP-(MIs)48 under normoxic conditions. (B) Size distribution of AL-PLGA/DOX, ALP-(MIs)25/DOX, and ALP-(MIs)48/DOX under normoxic conditions. (C) The stability of ALP-(MIs)25, ALP-(MIs)48, AL-PLGA, ALP-(MIs)25/DOX, ALP-(MIs)48/DOX and AL-PLGA/DOX NPs were investigated in DMEM containing 10% FBS over six days. (D-F) Size distribution of AL-PLGA, ALP-(MIs)25, and ALP-(MIs)48 under normoxic and hypoxic conditions. (G) The appearance change of AL-PLGA/DOX, ALP-(MIs)25/DOX, and ALP-(MIs)48/DOX under hypoxic conditions. (H) TEM images of ALP-(MIs)25 and AL-PLGA under normoxic and under hypoxic conditions. (I) Absorption spectra of ALP-(MIs)25, ALP-(MIs)48 and AL-PLGA when incubated under hypoxic and normoxic conditions for 2 h. (J) Hypoxic-responsive drug release from AL-PLGA/DOX, ALP-(MIs)25/DOX, and ALP-(MIs)48/DOX.

Figure 2

(A) Intracellular release of DOX from LP-(MIs)25/DOX and ALP-(MIs)25/DOX. Samples were incubated with C6 cells under normoxic and hypoxic conditions for 4 h. Scale bar: 50 μm. (B) Cellular uptake of LP-(MIs)25/DOX and ALP-(MIs)25/DOX was analyzed using flow cytometry after 4 h incubation under normoxic and hypoxic conditions. (C) Intracellular release of DOX from AL-PLGA/DOX and ALP-(MIs)25/DOX. Samples were incubated with C6 cells under normoxic and hypoxic conditions for 4 h. Scale bar, 50 μm. (D) Cellular uptake of AL-PLGA/DOX and ALP-(MIs)25/DOX was analyzed using flow cytometry after 4 h incubation under normoxic and hypoxic conditions.

Figure 3

Radiosensitization by ALP-(MIs)n in vitro. (A) Representative images of clonogenic survival assays of C6 cells cultured with AL-PLGA, ALP-(MIs)25, and ALP-(MIs)48 under hypoxic condition (pO₂: 2%) and ALP-(MIs)25 under normoxic condition following treatment with 0, 2, 4, 6 and 8 Gy. (B) Clonogenic survival curves of C6 cells cultured with AL-PLGA, ALP-(MIs)25, and ALP-(MIs)48 under hypoxic condition (pO₂: 2%) and ALP-(MIs)25 under normoxic condition following treatment with 0, 2, 4, 6 and 8 Gy. (C) Immunocytochemical analysis of γ-H2AX expressed by C6 cells. Cells were treated by incubation with AL-PLGA, ALP-(MIs)25, and ALP-(MIs)48 for 4 h under hypoxic condition (pO₂: 2%) and ALP-(MIs)25 under normoxic condition followed by irradiation with 2 Gy using a dose rate of 0.3 Gy min^-1. Cells were stained with an anti-γ-H2AX antibody (red) and DAPI (blue) 24 h after RT. Scale bar, 100 μm. (D) Quantitation of percentage of γ-H2AX positive cells. (Mean ± SD, n = 5, **p < 0.01).

Figure 4

DOX glioma distribution 24 h after intravenous injection of mice with either free DOX, LP-(MIs)25/DOX, LP-(MIs)48/DOX, ALP-(MIs)25/DOX, ALP-(MIs)48/DOX, and AL-PLGA/DOX. (A) Bioluminescence of luciferase-expressing tumor cells 10 min after injection with luciferin solution and fluorescence images of excised mouse brains. (B) Quantitative analysis of DOX fluorescence intensity in excised mouse brains. (Mean ± SD, n = 4, *p < 0.05) (C) Mean DOX concentration in excised mouse brains. (Mean ± SD, n = 4, *p < 0.05)

Figure 5

(A) Fluorescence images of free DOX, ALP-(MIs)25/DOX and ALP-(MIs)48/DOX tissue distribution (at a DOX concentration of 3 mg kg^-1) in intravenously injected C6-GFP-Luci-bearing ICR mice. (B) Quantitative analysis of DOX in tissues. (C) Blood retention kinetics of free DOX, ALP-(MIs)25/DOX and ALP-(MIs)48/DOX in ICR mice (DOX concentration of 3 mg kg^-1). All the data of blood retention kinetics were analyzed by Drug and Statistics (DAS) 3.23 software (mean ± SD, n = 4).

Figure 6

In vivo efficacy in the C6-GFP-Luci glioma mouse model. (A) C6-GFP-Luci-bearing mice received three injections of PBS, PBS + RT, ALP-(MIs)25/DOX, ALP-(MIs)48/DOX, LP-(MIs)25/DOX + RT, LP-(MIs)48/DOX + RT, ALP-(MIs)25/DOX + RT, and ALP-(MIs)48/DOX + RT at a dose of 3 mg kg^-1 DOX and 14.1 mg kg^-1 P-(MIs) on days 12, 14, and 16, with 2 Gy RT. (B) Bioluminescence signal change correlating to tumor growth over time following inoculation. (C) Quantification of the tumor bioluminescence signal. (n = 5 mice per group); *p < 0.05, ***p < 0.001, one-way ANOVA; n.s. indicates no statistical significance. (D) Kaplan-Meier survival curves for the mice (n = 10). *p < 0.05, one-way ANOVA. (E) Body weight change. Data are presented as the mean ± SD.

Figure 7

In vivo efficacy in the C6-GFP-Luci glioma mouse model. (A) C6-GFP-Luci-bearing mice received three injections of PBS, PBS + RT, free DOX + RT, AL-PLGA + RT, ALP-(MIs)25 + RT, ALP-(MIs)48 + RT, AL-PLGA/DOX + RT group, ALP-(MIs)25/DOX + RT and ALP-(MIs)48/DOX + RT at a dose of 3 mg kg^-1 DOX and 14.1 mg kg^-1 P-(MIs) on days 12, 14, and 16, with 2 Gy RT. (B) Bioluminescence signal change correlating to tumor growth over time following inoculation. (C) Quantification of the tumor bioluminescence signal (n = 5 mice per group); ** p < 0.01, one-way ANOVA; n.s. indicates no statistical significance. (D) Relative tumor inhibitory rate for each treatment; ** p < 0.01, one-way ANOVA; n.s. indicates no statistical significance. (E) Representative H&E-stained images of coronal sections from mouse brains with orthotopic tumors. (F) Ki67 staining of coronal sections from mouse brains with orthotopic tumors. Scale bar, 50 µm. (G) TUNEL staining of coronal sections from mouse brains with orthotopic tumors. Scale bar, 50 µm. (H) Kaplan-Meier survival curves for the mice (n = 10), *** p < 0.001, one-way ANOVA. (I) Body weight change. Data are presented as the mean ± SD.