(T1530-12-83) Development of a Novel Lipid Nanoparticle Based Immunochemotherapy against Brain Cancer

Purpose: Glioblastoma multiforme (GBM) is the most lethal and common primary brain malignancy. Despite the aggressive treatments, tumor recurrence is inevitable and hence a bleak prognosis with a median life expectancy of less than two years. To address the highly unmet medical need in the GBM therapy, the present research aims to develop a novel localized immunochemotherapy against GBM based on lipid nanoparticles (LNP). It is hypothesized that programing pro-tumor and immune-suppressive glioma-associated microglia/macrophages (M2-type GAM) in combination with targeted chemotherapy against glioma cells can augment GBM treatment.

Methods: BLZ945 and doxorubicin (DOX) were chosen as the model immuno- and chemotherapeutic, respectively. BLZ945 was encapsulated in mannose-modified LNP (BLZ-M-lips) that was previously optimized to possess the targeting ability to immune-suppressive M2-type macrophages. The LNP with the ability to “escape” from macrophages and target to glioma cells was used to load DOX (nanodisc-DOX). BLZ-M-lips and nanodisc-DOX were fabricated using a thin-film hydration method, separately. Drug loading and encapsulation efficiency were assessed by lysing LNP followed by HPLC analysis. The particle size and surface charge of the LNP were determined using Zetasizer. The polarization effect of BLZ-M-lips as well as in vitro anti-GBM effect of the proposed immunochemotherapy were evaluated using a co-culture model of Raw264.7 and GL261 cells.

Results: The mannose-modified lipid was successfully synthesized as confirmed by ¹H-NMR. BLZ-M-lips had a particle size of ~150 nm with polydispersity index (PDI) smaller than 0.1, which was confirmed by cryogenic transmission electron microscope (cryo-TEM) imaging (Fig. 1). Over 90% of BLZ-945 was encapsulated in BLZ-M-lips. The nanodisc-DOX has also been successfully developed and characterized (size: 39.5 ± 0.53 nm, volume%: >98%, encapsulation efficiency: >86%). In vitro cell study showed that free BLZ945 and BLZ-M-lips resulted in a higher M1/M2 phenotype ratio compared to non-treated control and non-targeted liposomal BLZ (BLZ-lips) groups in a Transwell co-culture model of Raw264.7 and GL261 (Fig. 2A). The immunochemotherapy based on the combinations of free BLZ945/DOX solutions and BLZ-M-lips/nanodisc-DOX demonstrated improved antitumor effect compared to the combinations of BLZ-lips and non-targeted liposomal DOX (DOX-lips) in vitro (Fig. 2B).

Conclusion: A novel immunochemotherapy based on the unique combination of BLZ-M-lips that targets M2-type GAM and nanodisc-DOX with superb exocytosis ability from M1-type GAM for sustained exposure of DOX to infiltrating glioma cells was developed. The BLZ-M-lips/nanodisc-DOX combination demonstrated synergistic effect against GBM. These findings have significant clinical implications and support preclinical studies of the combinational nanosized immune- and chemo- therapeutics to locally treat GBM, which remains a highly unmet medical need.

Figure 1. (A) Average particle size of the BLZ-M-lips was 148.8 ± 3.44 nm and PDI was 0.093 ± 0.053 using a Malvern Zetasizer (Mean±SD, n=3). (B) Cryo-transmission electron microscope (cryo-TEM) image of BLZ-M-lips.

Figure 2. (A) Fluorescence-activated cell sorting (FACS) was performed on Raw264.7 murine macrophages to determine the polarization ability of BLZ945 formulations (Mean±SD, n=3). (B) MTT assay was performed on GL261 murine glioma cells to determine the cell viability of BLZ and DOX formulations (Mean±SD, n=3). Statistical analyses were performed using Student’s t-test: **p < 0.01 and ****p < 0.0001.