Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo

Abstract

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

Keywords: DNA; brain tumor; gene therapy; nanomedicine; nonviral gene delivery; polymer.

Scheme 1. Schematic representation of the in vivo study. The 9L bearing rats were treated with intraperitoneal administration of ganciclovir twice a day beginning on day 4 and then treated with a single CED infusion of PBAE/HSV-tk nanoparticles on day 6 (A). These treated animals showed a significant benefit in survival (p = 0.0012 vs control) (B–D).

Figure 1

Polymer synthesis scheme and monomer chemical structures. Base monomers (B) and side chain monomers (S) are polymerized, and polymers are then end-capped with end-capping monomers (E).

Figure 2

PBAE nanoparticles effectively transfect 9L and F98 malignant glioma cells in vitro. All polymers were screened at 30, 60, and 90 (w/w) delivering 0.6 μg of GFP DNA (A and B). Of the nanoparticles tested on 9L and F98 cells, three and 15 formulations, respectively, were found to deliver GFP DNA more effectively than commercially available transfection reagent Lipofectamine 2000 (*p < 0.05 versus Lipofectamine with 0.6 μg DNA via one-way ANOVA with Dunnett’s post-test). Fluorescence microscopy shows cells transfected with GFP using PBAE nanoparticles (C and D). Transfection efficacy was quantified using flow cytometry. Loss in metabolic activity was quantified using an MTS assay with colorimetric readout, measured by a multiplate reader.

Figure 3

PBAE delivery of HSVtk plasmid enables GCV-mediated killing of malignant glioma cells in vitro. 9L and F98 cells were transfected with plasmids encoding either GFP or HSVtk and then treated with 0, 5, or 50 μg/mL GCV prodrug. Cells treated with both HSVtk and GCV exhibit 100% cancer cell killing, measured by cell counting, versus GFP-transfected cells treated with GCV, showing that GCV-induced cell killing is dependent on the presence of HSVtk. Additionally, HSVtk without GCV does not kill cells.

Figure 4

Nanoparticles maintain their physical characteristics and transfection capability following lyophilization. Fresh and lyophilized nanoparticles showed no statistical difference (p > 0.05) in their sizes and zeta potentials (A) and showed no statistical difference in percent transfection and geometric mean GFP in 9L cells (B). TEM imaging of fresh (C) and lyophilized (D) nanoparticles shows nanoparticles of the same size and morphology (scale bar = 100 nm).

Figure 5

Local brain delivery of PBAE/GFP Nanoparticle via CED leads to effective tumor transfection in vivo. Coronal section of a 9L tumor bearing rat brain at 7 days post PBAE/GFP infusion showing the tumor region (A, scale bar = 2 mm). Fluorescence microscopy images show GFP+ transfected cells in the tumor area (B, scale bar = 2 mm). Enlarged area shows a wide distribution of GFP+ cells within the entire tumor area including the periphery (C, scale bar = 500 μm). The colocalization of GFP and Cy5 shows that the nanoparticles penetrate into the cells and successfully transfect them (D–F, scale bar: 50 μm). Re,: Cy5; green, GFP; blue, DAPI.

Figure 6

PBAE/HSVtk nanoparticles and ganciclovir (GCV) extend survival in a 9L glioasarcoma model. Kaplan–Meier plots of F344 rats that were implanted with 9L and either given no treatment (9L Control, n = 16); 50 mg/kg twice a day of systemic administration of GCV on days 4–13 (GCV Alone, n = 8); intracranial infusion of PBAE/GFP nanoparticles plus systemic administration of GCV (NP-GFP + GCV, n = 8); intracranial infusion of HSVtk DNA plus systemic administration of GCV (DNA + GCV n = 8); or intracranial infusion of PBAE/HSVtk nanoparticles plus systemic administration of GCV (NP-HSVtk + GCV, n = 8). The median survival of the group receiving PBAE/HSVtk nanoparticles in combination with GCV is significantly longer compared to that of the untreated control group (p = 0.0012).