Harnessing Layer-by-Layer Assembly for Enhanced Nanoparticle Penetration and Glioblastoma Targeting in the Brain

Introduction:: Glioblastoma recurrence and ultimate mortality are often linked to cancer cell infiltration into regions with an intact blood-brain barrier, limiting the efficacy of systemic drug delivery. Convection-enhanced delivery (CED) offers an alternate method to directly administer therapeutics into the brain parenchyma, but its effectiveness is limited by rapid clearance and off-target uptake. Nanoparticle (NP) encapsulation presents a promising strategy to extend the retention time of locally delivered therapies and enable specific targeting of glioblastoma cells. However, the brain extracellular space poses challenges to nanoparticle distribution due to its narrow, tortuous pores and harsh ionic environment. Hydrophilic polymer coatings, such as polyethylene glycol (PEG), can provide colloidal stability and reduce non-specific interactions with the extracellular matrix (ECM). On the other hand, these antifouling coatings limit nanoparticle recognition by cancer cells, preventing the internalization of their payload. There remains a need to investigate surface chemistries that balance the competing considerations of extracellular distribution and glioblastoma-specific cellular targeting.


Materials and Methods:: We systematically investigated the impact of nanoparticle surface chemistry using layer-by-layer (LbL) assembly. Starting with fluorescently tagged anionic liposomes as a core substrate, we prepared a library of nanoparticles featuring 22 distinct outer-layer polyanions. We assessed glioblastoma cell uptake and specificity through a flow cytometry assay, utilizing a panel of patient-derived glioblastoma and primary healthy neural cells. To enable in vivo visualization of nanoparticle distribution in the brain, we incorporated a gadolinium-chelated lipid in the liposome core for MRI contrast. Following convection-enhanced delivery in both healthy and tumour-bearing mice, we evaluated the distribution and retention of nanoparticle formulations. Lastly, we analyzed cellular interactions with nanoparticles in vivo by dissociating mouse brains and conducting flow cytometry. This approach allowed us to quantify the nanoparticle content in seven major cell types, including tumor cells, providing insights into cellular fate and paving the way for the development of targeted therapies.


Results, Conclusions, and Discussions:: Our NP-cell interaction screens identified poly-L-glutamate (PLE) and sodium hyaluronate (HA) as outer layers with high affinity and specificity for patient-derived glioblastoma stem cells in vitro. Co-adsorbing either outer layer with a small fraction of PEGylated polyelectrolytes ensured sufficient colloidal stability in physiological conditions without sacrificing cancer cell affinity or specificity. Notably, this co-layering strategy improved the nanoparticle penetration of 3D glioblastoma spheroids. Following CED in both healthy and tumour-bearing mice, LbL-functionalized PLE and HA NPs exhibited an average volume of distribution three times larger than non-functionalized liposomes. LbL NPs demonstrated a mean retention half-time of 15 days, twice that of non-functionalized liposomes, indicating potential for long-acting, single-dose administrations. Flow cytometric analysis of dissociated mouse brains showed that LbL NPs increased uptake by cancer cells while reducing the uptake by healthy cells. Further examination of cellular interactions unveiled distinct cellular outcomes of PLE and HA LbL NPs among tumour and immune cells, offering opportunities to tailor this system for various therapeutic applications. Upcoming studies will evaluate the therapeutic efficacy of locally delivered drug-loaded LbL NPs for glioblastoma treatment in vivo.


Acknowledgements (Optional): : The authors would like to thank the Koch Institute's Robert A. Swanson (1969) Biotechnology Center for technical support, specifically the Preclinical Modeling and Testing Core, Flow Cytometry Core, and Microscopy Core. We would also like to acknowledge funding from Cancer Research UK and the Brain Tumour Charity grant REF: C42454/A28596. A.J.P. is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), [CGSD3 - 557538 - 2021].

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