(W1130-11-75) Tailoring Ligand-Altered Nanostructured Lipid Carrier for the Brain Delivery of Anti-cancer Drug cum herbal Compound for the Treatment of Glioblastoma Multiforme

Purpose: Glioblastoma multiform (GBM) is the most lethal form of brain cancer pigeonholed by the high rate of reoccurrence and poor diagnosis. Among all treatment strategies available, chemotherapy is considered the most common treatment entity at a clinical level. An anti-mitotic taxane drug (TX), shows its cytotoxic and anti-proliferative effect by modulating microtubule polymerization, decreasing depolymerization and arresting cells at the G1/M phase of the cancerous cell cycle. Despite being highly effective in treating solid brain tumor, it possesses various limitations namely hydrophobicity, dose-dependent cytotoxicity and sensitivity towards P-glycoprotein (P-gp) pumps. A natural flavonoid (FV) possesses anti-oxidant, anti-inflammatory property and also acts as a P-gp inhibitor. FV has potential in inducing apoptosis, along with arresting cells in the G1/M phase of the cell cycle providing apoptotic and anti-proliferative effects. Since both the drugs belong to BCS class IV (low solubility and permeability) therefore, their accumulation at tumor site is limited. Thus, there comes a need to increase the dose of the drugs to achieve a significant anti-tumor effect in the brain. So, this study focuses on the development of a nanostructured lipid carrier (NLC) loaded with the combination of TX and FV to achieve their synergistic effect at tumor site i.e., the brain. Furthermore, the specificity and performance of TX-FV NLC will be improved by conjugating it with the ligand. The selection of ligands has been done based on localised receptors at tumor site. As it will help in enhancing drug targeting efficiency of NLC thereby causing minimal peripheral drug toxicity and maximum apoptotic and anti-proliferative effect. Administration of the developed formulation via intranasal route will deliver this combination at tumor site following trigeminal and olfactory pathways.

Methods: TX-FV loaded NLC was developed by a modified emulsio-sonication (fusion of emulsification and sonication) method followed by optimization using a Central composite design. Later this formulation was conjugated with a ligand which was then evaluated for its globule size, polydispersity index (PDI), zeta potential (ZP) and entrapment efficiency (EE). Different methods including scanning electron microscopic (SEM) and Transmission electron microscopy (TEM) were used to appraise the morphology of TX-FV NLC and ligand-conjugated TX-FV NLC. Moreover, the crystallinity of each drug and ligand in the NLC was examined by powdered differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The ligand-conjugated optimized NLC was then assessed for its release profile by performing in-vitro and ex-vivo studies in comparison to the TX-FV suspension, and TX-FV NLC. Well along, Confocal laser scanning microscopy (CLSM) was used to evaluate the permeation potential amongst these formulations.

Results: TX-FV loaded NLC was successfully formulated using a modified emulsio-sonication method followed by conjugating with ligand, later optimized grounded on the physicochemical features and stability. The ligand-conjugated TX-FV NLC possessed a globule size of (197.53 – 208.68 nm), polydispersity index (0.309 – 0.336), zeta potential (-14.26 to -15.78 mV) and entrapment efficiency of TX (82.36 ± 2.67 %); FV (84.45 ± 2.04%). The TEM and SEM results showed that the developed formulation was spherical. The DSC and XRD didn’t show characteristic peaks of individual drugs and ligand in the formulation due to the dispersion of these drugs in the lipids. Alternatively, the comparative in-vitro study amongst different formulations showed initial burst release followed by sustained release of TX and FV from NLC and ligand-conjugated NLC in comparison to suspension. Whereas, ex-vivo permeation using goat nasal mucosa showed a 2-3fold increase in the flux of TX and FV from NLC and ligand-conjugated NLC in comparison to suspension. In addition, the confocal study showed significant permeation of drug combination across nasal mucosa from formulations (20-25µm) in comparison to that suspension (15µm) demonstrating excellent in vivo prospect of formulation.

Conclusion: Based on the results obtained, it can be well predicted that NLC and ligand-conjugated NLC had great potential in circumventing the problems associated with recent treatment strategies for the treatment of GBM. The in-vitro and ex-vivo studies demonstrated that the developed NLC and ligand-conjugated NLC meaningfully enhanced the release and permeation of the drugs. To conclude study findings established that the developed ligand-conjugated TX-FV NLC possesses great potential for enhancing the accessibility and accumulation of chemotherapeutic agents at the tumor site and thereby, substantially improving the therapeutic efficacy.

References: Anjum K, Shagufta BI, Abbas SQ, Patel S, Khan I, Shah SAA, et al. Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review. Biomed Pharmacother. 2017;92:681–9.
Li AJ, Zheng YH, Liu GD, Liu WS, Cao PC, Bu ZF. Efficient delivery of docetaxel for the treatment of brain tumors by cyclic RGD-tagged polymeric micelles. Molecular medicine reports. 2015 Apr 1;11(4):3078-86
Kim H, Moon JY, Ahn KS, Cho SK. Quercetin induces mitochondrial-mediated apoptosis and protective autophagy in human glioblastoma U373MG cells. Oxidative Medicine and Cellular Longevity. 2013 Oct;2013.
Zhai K, Mazurakova A, Koklesova L, Kubatka P, Büsselberg D. Flavonoids synergistically enhance the anti-glioblastoma effects of chemotherapeutic drugs. Biomolecules. 2021 Dec;11(12):1841.
Acknowledgements: The authors are thankful for the DST PURSE and DST FIST grant to the Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India

Schematic illustration of the targeted delivery of ligand conjugated dual drug-loaded NLC formulation to the tumor cells for the treatment of GBM via intranasal route