Introduction: Glioblastoma multiforme (GBM) is one of the most aggressive and lethal forms of brain cancer, characterized by rapid proliferation, diffuse infiltration, and resistance to conventional therapies. A major challenge in treating GBM is the presence of the blood-brain barrier (BBB), which limits the delivery of therapeutic agents to the tumor site. Additionally, the loss of PTEN, a critical tumor suppressor gene, is a common mutation in GBM, leading to uncontrolled cell growth and survival. Restoring PTEN function through gene therapy presents a promising strategy, but effective delivery systems are crucial for its success.
This study aims to address these challenges by developing a nanoparticle-based delivery system for PTEN mRNA. The nanoparticles are engineered with a poly(lactic-co-glycolic acid) (PLGA) core for sustained release of the mRNA and a poly(ethylene glycol)-b-poly(aspartic acid) (PEG) shell that is pH-responsive, degrading in the acidic tumor microenvironment to release the mRNA specifically at the tumor site. The nanoparticles are further coated with erythrocyte membranes (EMs) to evade the immune system, prolonging circulation time, and are functionalized with Apolipoprotein E (ApoE4) peptides to enhance BBB penetration.
To monitor the success of the delivery, a pCMV-GFP plasmid encoding Green Fluorescent Protein (GFP) is co-encapsulated with the PTEN mRNA, allowing for visualization of transfection efficiency. This approach is designed to restore PTEN expression in GBM cells, thereby inhibiting tumor growth and providing a foundation for further development of mRNA-based therapies for GBM.
Materials and
Methods: The nanoparticle system was designed for targeted delivery of PTEN mRNA to glioblastoma cells. Nanoparticles were synthesized with a poly(lactic-co-glycolic acid) (PLGA) core to encapsulate PTEN mRNA, providing controlled release. Surrounding the core was a poly(ethylene glycol)-b-poly(aspartic acid) (PEG) shell, engineered to degrade in the acidic environment typical of the glioblastoma tumor microenvironment (pH 5.0-6.5), thus ensuring selective release of PTEN mRNA at the tumor site. The nanoparticles were coated with erythrocyte membranes (EMs) to enhance their circulation time and immune evasion.
To facilitate penetration across the blood-brain barrier (BBB), the nanoparticles were functionalized with Apolipoprotein E (ApoE4) peptides, specifically the 142-150 fragment known for its BBB-crossing abilities. U87MG glioblastoma cells, cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, were used for in vitro transfection experiments. PTEN mRNA-loaded nanoparticles were introduced to the cells using Lipofectamine 3000, a widely-used transfection reagent.
To track transfection efficiency, GFP mRNA was co-encapsulated within the nanoparticles, with GFP fluorescence serving as an indicator of successful mRNA delivery and expression. PTEN protein expression was subsequently quantified using Western blot analysis. The therapeutic efficacy of the PTEN expression was evaluated through BrdU incorporation assays to measure cell proliferation. Additionally, an in vitro BBB model was employed using Transwell chambers, allowing assessment of the nanoparticles' ability to cross the BBB and deliver mRNA to glioblastoma cells.
Results, Conclusions, and Discussions: Our results demonstrated the successful transfection of U87MG glioblastoma cells with the engineered nanoparticles, as evidenced by GFP fluorescence, which confirmed efficient delivery and expression of PTEN mRNA. The pH-responsive PEG shell was instrumental in achieving targeted release, degrading in the acidic tumor microenvironment to release the mRNA payload precisely where it was needed. Western blot analysis corroborated the successful translation of PTEN mRNA, showing a significant increase in PTEN protein levels. This was further supported by BrdU incorporation assays, which indicated a marked reduction in cell proliferation among treated cells, highlighting effective inhibition of tumor growth. Additionally, flow cytometry revealed an increase in apoptotic cells following treatment, suggesting that restored PTEN function enhanced programmed cell death, contributing to the suppression of glioblastoma. These findings underscore the efficacy of the engineered nanoparticles in not only delivering but also activating PTEN within the tumor cells. The in vitro BBB model provided further validation of the system’s potential, demonstrating that the ApoE4-functionalized nanoparticles could cross the blood-brain barrier and deliver PTEN mRNA to cells situated beyond the barrier. This success in crossing the BBB addresses one of the significant challenges in brain tumor therapy, highlighting the promising nature of this approach. In the future, this study underscores the potential of mRNA-based nanoparticle therapies for treating glioblastoma. The combination of a pH-responsive release mechanism, immune-evasive coatings, and BBB-penetrating peptides positions this strategy as a viable solution for overcoming current therapeutic limitations. Future research should focus on in vivo studies to further evaluate the clinical efficacy and safety of this delivery system. Additionally, exploring combination therapies and optimizing nanoparticle formulations could enhance therapeutic outcomes and facilitate the transition of this technology from the lab to clinical settings. This approach could significantly advance glioblastoma treatment and potentially be adapted for other challenging cancers.
Acknowledgements (Optional): Thank you to the Stanford iGEM Bioengineering Research team for facilitating this proposal!
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