Glioblastoma multiforme (GBM) is the most aggressive and commonly diag- 11 nosed brain cancer and presents a strong resistance to routine chemotherapeutic drugs. 12 The present study involves the synthesis of Lignin-g- p (NIPAM-co-DMAEMA) gold 13 nanogel, loaded with curcumin and piperine to treat GBM. The application has three 14 functions: (1) overcome the limitations of biodistribution, (2) enhance the toxicity of an- 15 ticancer drugs against GBM, (3) identify the uptake pathway. Atom transfer radical 16 polymerization was used to synthesize the Lignin-g-PNIPAM network, crosslinked with 17 the gold nanoparticles (GNPs) to self-assemble into nanogels. The size distribution and 18 morphological analysis confirmed that the drug-loaded gold nanogels are spherical and 19 exist in the size of 180 nm. The single and combinatorial toxicity effects of curcumin and 20 piperine loaded Lignin-g- p (NIPAM-co-DMAEMA) gold nanogels were studied against 21 GBM cells. A cytotoxicity analysis against glioblastoma cells (U-251 MG) displayed an- 22 ticancer properties. IC50 of curcumin and piperine-loaded gold nanogels were recorded 23 at 30 μM and 35 μM respectively. Immunostaining analysis of the drug-loaded nanogel 24 treated cells shows that the F-actin induced cytoskeletal deformations result in the trig- 25 gering of caspase-3 apoptotic pathways. Kinetic drug release revealed the 86% release of 26 hybrid curcumin-piperine from nanogel after 250 mins at pH 4. Atomic absorption 27 spectroscopic analysis confirmed that the drug-loaded nanogels have better internaliza- 28 tion or association with the cancer cells than the GNPs or nanogels alone. Morphology 29 studies further confirmed that the curcumin and piperine nanogels penetrate the cells via 30 endocytic pathways and induce caspase-3 related apoptosis. The experimental evidence 31 shows the enhanced synergistic properties of combinatorial curcumin-piperine gold 32 nanogels (IC 50 : 21 μM) to overcome the limitations of conventional chemotherapeutic 33 treatments of glioma cells
