Optimal strategies for Convection Enhanced Delivery (CED) of DNA Brain‐Penetrating Nanoparticle (DNA‐BPN) for Glioblastoma (GBM) Treatment

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Although gene therapy has shown a powerful strategy for GBM treatment in preclinical studies, its effectiveness has yet to be achieved in humans due in large part to an inability to achieve widespread distribution in the brain and tumor selectivity. Direct infusion into the tumor tissue is the most straightforward approach to bypass the blood brain barrier (BBB), which is the primary hurdle experienced by systemically administered therapeutics, including gene vectors. Specifically, intracranial convection enhanced delivery (CED) provides a pressure‐driven flow that facilitates distribution of gene vectors away from the point of administration. However, the nano‐porous and highly adhesive extracellular matrix (ECM) effectively traps therapeutic nanoparticles (NP) via multivalent adhesive interactions and/or steric obstruction. NP trapped at the injection site cannot flow through the brain parenchyma and distribute away from the point of administration, limiting the potential for clinical success; in the case of gliomas, invasive cancer cells that migrate away from the bulk of the tumor mass into the healthy brain parenchyma are difficult to access, and thus, are primarily responsible for tumor relapse. We recently demonstrated that the key to achieve NP penetration within the brain is to engineer particles possessing a non‐adhesive particle surface which is achieved by dense surface coatings with hydrophilic and neutrally charged polyethylene glycol (PEG). With this lesson, we have developed methods to formulate DNA nanoparticles (DNA‐NP) capable of rapidly penetrating brain tissues (i.e. brain‐penetrating NP; DNA‐BPN), using a blend of cationic polymers and PEG‐conjugated cationic polymers; PEGylated polymers possess a range of molar ratios of PEG and cationic polymers. We further went on to determine an optimal PEG density that provides the highest level of transfection in the brain. Specifically, we compared DNA‐NP formulated with PEGylated polymers possessing varying PEG to cationic polymer ratios. Our data shows that DNA‐NP formulated with an inclusion of PEGylated polymers at the highest PEG content forms the most stable DNA‐BPN, retaining their physiocochemical properties in physiologically relevant conditions. Following CED, the lead DNA‐BPN provided markedly greater distribution and overall levels of transgene expression in the brain compared to DNA‐BPN formulations with lower PEG contents. We also optimized the infusate solution to further enhance the distribution of the lead DNA‐BPN. To ensure that our formulation can be stored long term for clinical use, we tested the effect of lyoprotection on the aforementioned properties and performances of the lead DNA‐BPN, using different commonly used disaccharide lyoprotectants. Our future work will involve an incorporation of tumor‐specific promoters to achieve widespread but cancer‐selective therapeutic gene transfer for highly effective and safe glioma gene therapy. Support or Funding Information Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (Parent F31) FCA210610A NIH R01CA204968 NIH R01EB0201471 Physiochemical properties and colloidal stability of gene vectors with PEI polymer.Hydrodynamic diameter ± SEM (nm) ζ‐Potential ± SEM (mV) PDIDNA‐CPN 49.5 ± 0.9 7.6 ± 0.2 0.16 DNA‐BPN‐30 48.5 ± 1.1 4.0 ± 0.3 0.19 DNA‐BPN‐50 48.1 ± 5.2 2.0 ± 0.1 0.202 Sizes of PEGylated polystyrene (PS‐PEG) and UN‐PEGylated polystyrene (PS‐COOH) nanoparticles hypotonic, isotonic, or hypertonic saline solutions.Infusate Solution Water 0.9% Saline 3% SalineOsmolality (mOsm/kg) 0 ~300 ~1000Viscosity 0.89 0.90 0.94PSPEG (nm) 58 ± 0.2 61 ± 2 62 ± 0.5PSCOOH (nm) 51 ± 1 45 ± 2 230 ± 57