Comparative Efficacy of Tumor Microenvironment-responsive Nanotherapeutics Targeting PSD95/Discs-large/ZO-1 Binding
Qi Zhang1, Chao Hu2, Baoqing Qu1
1Department of Ultrastructural Pathology, Beijing Neurosurgical Institute/ Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070, China.
Abstract:
This work aimed to demonstrate the therapeutic effects of tumor microenvironment-responsive nanotherapeutic drugs targeting PSD95/Discs-large/ZO-1 domain (PDZ)-binding-kinase (PBK) in medulloblastoma Daoy and ONS-76 cells. The objective was to provide critical theoretical and practical foundations for the clinical adoption of tumor microenvironment-responsive nanotherapeutic drugs targeting PBK. The rabies virus glycoprotein (RVG) was utilized as a specific targeting molecule to form a tumor microenvironment-responsive nanocomplex, HPAA/RVG/PBK-siRNA, which incorporated glutathione (GSH) as a microenvironment stimulus factor within a hyperbranched polymer polyamide amine (HPAA). This nanocomplex also carried PBK-small interfering RNA (siRNA) for targeted PBK therapy. Characterization of HPAA, maleimide-polyethylene glycol-N-succinyl ester (MAL-PEG-NHS), HPAA-PEG, RVG, HPAA-RVG, and HPAA/RVG/PBK-siRNA was conducted using nuclear magnetic resonance spectroscopy, high-performance liquid chromatography (HPLC), dynamic light scattering, and transmission electron microscopy (TEM). Flow cytometry was employed to assess endocytosis and cell transfection of HPAA-RVG and HPAA/RVG/PBK-siRNA in Daoy and ONS-76 cells. The two cell lines were treated with HPAA/RVG/PBK-siRNA (HPAA/siRNA group), methoxy-PEG polyethylenimine (PEI-25k)/PBK-siRNA (PEI group), HPAA/RVG nanocarriers without PBK-siRNA (HPAA/RVG group), Dharmacon™ non-targeting siRNA (shNTC group), PBK-siRNA (Control group 1), AChR inhibitor (Control group 2), and GSH inhibitor (Control group 3), and compared with the control group (medium without any substances). Western blot analysis validated PBK expression levels (ELs) in various cell groups. Additionally, cell viability and proliferation were evaluated using methyl tetrazolium (MTT) assays and 5-Bromo-2'-deoxyuridine (BrdU) incorporation assays. The results revealed proton absorption peaks for HPAA at 2.78 ppm, 3.21 ppm, and 3.49 ppm, while RVG and HPAA-RVG exhibited characteristic absorption peaks at 23.653 min and 23.584 min, respectively, with peak areas of 4,856.6 and 6,927.3 for RVG. The nanoparticle sizes were 50-100 nm for HPAA-RVG and 100 nm for HPAA/RVG/PBK-siRNA, displaying spherical morphology and uniform size distribution. The average potential of HPAA-PEG was lower than that of HPAA (P<0.05), and HPAA-RVG showed dramatically lower potential than HPAA (P<0.001). At 8 hours, Daoy cells displayed higher endocytosis rates versus ONS-76 cells (P<0.05). The transfection rates of HPAA-RVG in both ONS-76 and Daoy cells were higher than those of HPAA, with Daoy cells showing higher transfection rates than HPAA (P<0.05). Under HPAA-RVG treatment, AChR levels in ONS-76 cells were significantly lower than those in Daoy cells (P < 0.05). Compared to the control group, the PBK protein expression levels, cell survival rates, and the number of cells in the proliferative phase were significantly reduced in Control group 1, the PEI group, and the HPAA/siRNA group in both ONS-76 and Daoy cells, with the ONS-76 cells in the HPAA/siRNA group showing the lowest values among these groups (P < 0.05). In summary, the findings indicated that the tumor microenvironment-responsive nanocomposite HPAA/RVG/PBK-siRNA selectively inhibited PBK expression in Daoy medulloblastoma cells, showcasing potential applicability in medulloblastoma therapy.
Insights
This study developed a novel nanotherapeutic drug that targets PBK in medulloblastoma cells. The drug demonstrated significant therapeutic effects by reducing tumor cell viability and proliferation, offering a promising new treatment strategy.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Oncology
Background:
- Medulloblastoma is a common pediatric brain tumor with limited treatment options.
- Targeting the PBK pathway presents a potential therapeutic strategy.
- Developing effective drug delivery systems is crucial for cancer therapy.
Purpose of the Study:
- To develop and characterize a tumor microenvironment-responsive nanotherapeutic drug targeting PBK in medulloblastoma.
- To evaluate the therapeutic efficacy of the nanodrug in Daoy and ONS-76 medulloblastoma cell lines.
- To provide a foundation for the clinical application of PBK-targeted nanotherapeutics.
Main Methods:
- Synthesis and characterization of HPAA/RVG/PBK-siRNA nanocomplex using spectroscopy, HPLC, DLS, and TEM.
- Assessment of cellular uptake and transfection efficiency via flow cytometry.
- Evaluation of PBK expression, cell viability, and proliferation using Western blot, MTT, and BrdU assays.
Main Results:
- The HPAA/RVG/PBK-siRNA nanocomplex exhibited optimal nanoparticle size and morphology.
- The nanodrug demonstrated efficient cellular uptake and transfection in medulloblastoma cells.
- Significant reduction in PBK expression, cell viability, and proliferation was observed in treated cells, particularly in ONS-76 cells.
Conclusions:
- The developed tumor microenvironment-responsive nanotherapeutic drug effectively targets and inhibits PBK in medulloblastoma cells.
- The nanodrug shows significant therapeutic potential for medulloblastoma treatment.
- This study provides critical insights for the clinical translation of PBK-targeted nanotherapeutics.
