Glioblastoma (GB) is the most frequent and lethal primary brain tumor. GB has currently no cure and the standard care regimens only provide patients a median survival of 12-15 months after diagnosis. Indeed, considering the unfeasibility of performing complete surgical resection and the low efficacy of chemoradiotherapy in eliminating the remaining GB cells, it is not possible to circumvent tumour recurrence. Therefore, there is an urgent need to develop an efficient treatment for GB. This work proposes a hydrogel designed for the direct injection into the resection cavity, allowing starting the treatment immediately after surgery. Being locally administered, it can overcome possible problems related to e.g. systemic side effects and reduced brain penetration. Moreover, the viscoelastic behaviour of the matrix allows the desirable interaction and attachment of the surrounding cancer cells, without the detrimental effects of the tumor microenvironment. Thus, it simultaneously promotes the sustained release of drugs to efficiently damage GB cells while avoiding stimulatory extracellular matrix effects on tumour cells.
The hydrogel is based on hyaluronic acid (HA) functionalized with the fibronectin inhibitor peptide Arg-Gly-Asp-Ser (RGDS) and physically crosslinked with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) large unilamellar liposomes (LUVs) encapsulating doxorubicin (DOX). The peptide was synthesized in an automated peptide synthesizer, followed by its characterisation by HPLC-MS. Then, it was used to functionalize HA through carbodiimide chemistry. HA functionalization was confirmed by NMR and ATR-FTIR spectroscopy. LUVs were prepared by the thin-film hydration method followed by extrusion. LUVs’ characterisation included the determination of size distribution, surface charge, morphology, phase transition, stability and drug concentration. Hydrogels were characterized in terms of viscoelastic and thermal properties and structure. Drug release profile from hydrogels was evaluated by HPLC. The ability of the matrix metalloproteinase-2 (MMP-2) produced by GB cells to break the peptide-HA binding was also assessed. The human primary GB cell line GBML42 and astrocytes were used to assess the therapeutic value and safety of the developed formulation.
LUVs with DOX presented a homogeneous size of ≈121.7 nm and a slightly negative zeta potential (≈-2.43 mV). Moreover, DOX was encapsulated in relevant concentrations (≈68.2 µM in 1 mM LUVs) considering the DOX IC50 results (≈3.82 µM at 24 h of treatment). The hydrogel presented rheological properties similar to the healthy brain and was able to sustain the release of DOX. In vitro assays demonstrated the efficacy of unmodified HA hydrogels with liposomes encapsulating DOX to damage GB cells. Conversely, RGDS-functionalized HA hydrogels presented cytotoxicity even without DOX incorporation. Indeed, MMP-2 disrupted the peptide-HA bond. Thus, the internalization of free RGDS can lead to GB cells apoptosis. Importantly, RGDS-functionalized HA hydrogels incorporating liposomes with DOX efficiently damaged GB cells without affecting the metabolism and viability of astrocytes, proving their safety.
RGDS increased the cytotoxicity of the system, proving how it can act synergistically with the incorporated drug for GB treatment. Thus, this work shows the potential of this formulation to be used as a safe and effective local treatment for GB.
Acknowledgments: H2020 (668983-FORECAST), FCT (PTDC/BTMSAL/28882/2017–Cells4_IDs) and NORTE2020 (NORTE-01-0145-FEDER-000021).