(28l) Leveraging Synergy of Hitchhiking Nanocarriers and Chemotherapy to Overcome Delivery Challenges in Glioblastoma

Introduction: Glioblastoma Multiforme (GBM) is a heterogeneous, damaging and aggressive type of brain tumor that originates from abundant non-neuronal glial cells called astrocytes. A study of GBM cases show that 60% of the occurrences arise de novo and 40% progress from a lower-grade brain tumor. As per recent reports, the mean survival rate is only about 8 – 15 months for recently diagnosed GBM cases whereas it is 3 – 9 months for recurrent GBM. Regardless of aggressiveness, multimodal therapies on the majority of patients fail to attain a sustained response and ultimately succumb to the disease. All in all, the 5 – year rate of survival is observed in less than 10% of cases. Existing therapeutic strategies for the treatment of GBM include gross total or subtotal surgical resection, chemotherapy, external radiotherapy and concurrent therapy. However, its efficacy in vivo is limited owing to its neurologic (sensory, visual defects, motor weakness and language problem), regional (seizure, meningitis, cerebrospinal fluid leak and wound infection) and systemic adverse effects (pulmonary infection, thrombosis, psychosis and pulmonary embolism) that arise as long-term consequences of surgical resection. External radiation therapies are reported to entail long term effects pertaining to neurocognitive deterioration. Additionally, the gold standard chemotherapy – temozolomide is stable in the acidic microenvironment of the tumor interstitium and thereby does not transform into the DNA methylating species required for DNA damage, limiting its therapeutic potency. Furthermore, the inefficacy of chemotherapy-based treatment due to its heterogenous intratumoral diffusion and permeability of the blood brain tumor barrier combined with triggered cellular resistance pose challenges necessary to overcome.

We investigate the synergistic potential of RadioPharmaceutical Therapy (RPT), specifically α-particle emitters (α-RPT) with chemotherapy to treat glioblastoma. α-RPT, is a form of targeted internal radiotherapy that involves deposition of massive amounts of energy by highly charged α-particles as they traverse tissue and produce complex double-strand breaks in DNA that impair cellular repair in diseased tissue at proximity. With their short range of 5 – 10 cell diameters, α-particles allow for localized irradiation of target tumor cells with minimal toxicity to peripheral healthy cells without dependence on the oxygenation state of the cell or the cell cycle. Since the killing efficacy of α-particles is dictated by its ability to cause double-stranded DNA breaks, its ability to treat solid tumors is only limited by its short range. Furthermore, conventional therapeutic carriers (such as liposomes ~ 100nm in diameter, and/or antibodies) pose obstructed crossing of the blood brain tumor barrier and diffusion limited transport within the tumor, ultimately resulting in partial irradiation of the tumor.

Here, we investigate a novel delivery strategy for systemic administration that combines utilization of existing biological mechanisms to promote selective and uniform delivery of a cytotoxic agent (an α-particle emitter) loaded on dendrimer nanoparticles with temozolomide, a chemotherapeutic agent to achieve a synergistic effect aiming at reduced disease recurrence or resistance and prolonged survival. These dendrimers (7.5 – 10 nm) (1) can easily permeate the blood brain tumor barrier upon systemic administration owing to their nanosize, but do not permeate the blood brain barrier of the healthy tumor; (2) They can hitchhike tumor associated macrophages (GBM cells reprogram macrophages into tumor associated macrophages, which suppress antitumor immune activation) that infiltrate the tumor microenvironment, allowing for the α-emitters to uniformly irradiate brain tumors at the site of cancer-induced inflammation; and (3) They quickly clear from the body when not associated with tumor associated macrophages, thereby decreasing off-target toxicities.

Materials and Methods: In vitro, the uptake of dendrimers in comparison to large carriers (liposomes) by activated macrophages was determined. The sensitivity of GL261 cells to the free form of drug (in this case ²²⁵Ac-DOTA), ²²⁵Ac radiolabeled dendrimer nanoparticles and temozolomide at the physiological pH 7.4 and acidic pH 6.2 was evaluated using clonogenic survival assays. To assess the efficacy of the proposed therapeutic regime, 3D multicellular spheroids of glioblastoma GL261 cells (analogues of the tumor avascular regions) were developed and their ability to mimic the acidic tumor microenvironment was confirmed with measurement of their interstitial pHe profile. The extent of cell survival exposed to radiolabeled dendrimers and temozolomide as single and combination treatments were studied in the developed 3D tumor models. The interstitial microdistributions of cy5 labelled dendrimer and its time integrated radial concentrations were evaluated.

Results and Discussion: The uptake of dendrimers (Figure 1A) by activated macrophages was significantly higher than the uptake of larger drug carriers (liposomes). Clonogenic survival assays indicated lower cell survival with ²²⁵Ac radiolabeled dendrimer than ²²⁵Ac-DOTA (Figure 1B) and confirmed lower efficacy of temozolomide in the acidic tumor interstitium than at physiologic pH (Figure 1C). In GBM spheroids formed by the same GL261 cell line, the interstitial pHe acidification was measured (Figure 1D). The extent of spheroid outgrowth/regrowth inhibition after exposure to a combination of chemotherapy with α-particle therapy was more pronounced compared to each of the independent therapies alone (Figure 1E), independent of the interstitial acidification which was shown (Figure 1C) to limit the efficacy of temozolomide. Further experiments are planned to weight the importance of (a) the delivery (transport within the spheroids) (Figure 1F) and/or (2) the cancer cell biology, as a response to both agents, to the inhibition findings in 3D spheroids.

Conclusions: In this study, we conclude that the observed synergy in limiting spheroid regrowth treated with chemotherapy and α-particle-labeled dendrimers (which are expected to ultimately be infiltrated within tumors by hitchhiking the tumor associated macrophages) could potentially overcome diffusion limited transport within intracranial tumors and ultimately become a successful therapeutic intervention to improve life expectancy.