Glycan Modulation of Insulin-like Growth Factor-1 Receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) that plays critical roles in cancer. Microarray, computational, thermodynamic, and cellular imaging studies reveal that activation of IGF-1R by its cognate ligand IGF1 is inhibited by shorter, soluble heparan sulfate (HS) sequences (e.g., HS06), whereas longer polymeric chains do not inhibit the RTK, a phenomenon directly opposed to the traditional relationship known for GAG-protein systems. The inhibition arises from smaller oligosaccharides binding in a unique pocket in the IGF-1R ectodomain, which competes with the natural cognate ligand IGF1. This work presents a highly interesting observation on preferential and competing inhibition of IGF-1R by smaller sequences, whereas polysaccharides are devoid of this function. These insights will be of major value to glycobiologists and anti-cancer drug discoverers.

Inducing endoplasmic reticulum stress in cancer cells using graphene oxide-based nanoparticles.

The endoplasmic reticulum is one of the vital organelles primarily involved in protein synthesis, folding, and transport and lipid biosynthesis. However, in cancer cells its functions are dysregulated leading to ER stress. ER stress is now found to be closely associated with hallmarks of cancer and has subsequently emerged as an alluring target in cancer therapy. However, specific targeting of the ER in a cancer cell milieu remains a challenge. To address this, in this report we have engineered ER-targeted self-assembled 3D spherical graphene oxide nanoparticles (ER-GO-NPs) encompassing dual ER stress inducers, doxorubicin and cisplatin. DLS, FESEM and AFM techniques revealed that the nanoparticles were spherical in shape with a sub 200 nm diameter. Confocal microscopy confirmed the specific homing of these ER-GO-NPs into the subcellular ER within 3 h. A combination of gel electrophoresis, confocal microscopy and flow cytometry studies revealed that these ER-GO-NPs induced ER stress mediated apoptosis in HeLa cells. Interestingly, the nanoparticles also activated autophagy which was inhibited through the cocktail treatment with ER-GO-NPs and chloroquine (CQ). At the same time these ER-GO-NPs were found to be efficient in prompting ER stress associated apoptosis in breast, lung and drug resistant triple negative breast cancer cell lines as well. We envision that these ER specific self-assembled graphene oxide nanoparticles can serve as a platform to exploit ER stress and its associated unfolded protein response (UPR) as a target resulting in promising therapeutic outcomes in cancer therapy.

Supramolecular self-assembly of triazine-based small molecules: targeting the endoplasmic reticulum in cancer cells.

The endoplasmic reticulum (ER) is one of the most important organelles controlling myriads of cellular functions including protein folding/misfolding/unfolding, calcium ion homeostasis and lipid biosynthesis. Subsequently, due to its functional dysregulation in cancer cells, it has emerged as an interesting target for anti-cancer therapy. However, specific targeting of the ER in cancer cells remains a major challenge due to the lack of ER-selective chemical tools. Furthermore, for performing multiple cellular functions the ER is dependent on the nucleus through complicated cross-talk. Herein, we have engineered a supramolecular self-assembled hexameric rosette structure from two small molecules: tri-substituted triazine and 5-fluorouracil (5-FU). This rosette structure consists of an ER-targeting moiety with a fluorescence tag, an ER-stress inducer and a nuclear DNA damaging drug simultaneously, which further self-assembled into an ER-targeting spherical nano-scale particle (ER-NP). These ER-NPs internalized into HeLa cervical cancer cells by macropinocytosis and specifically localized into the ER to induce ER stress and DNA damage leading to cell death through apoptosis. Interestingly, ER-NPs initiated autophagy, inhibited by a combination of ER-NPs and chloroquine (CQ) to augment cancer cell death. This work has the potential to exploit the concept of supramolecular self-assembly into developing novel nano-scale materials for specific sub-cellular targeting of multiple organelles for future anti-cancer therapy.

Polyethylenimine Coated Graphene Oxide Nanoparticles for Targeting Mitochondria in Cancer Cells.

Mitochondrion, the powerhouse of the cells, controls bioenergetics, biosynthesis, metabolism, and signaling. Consequently, it has become an unorthodox target for cancer therapeutics. However, specific targeting of mitochondria into subcellular milieu in cancer cells remains a major challenge. To address this, we have engineered polyethylenimine cloaked positively charged self-assembled graphene oxide nanoparticle (PEI-GTC-NP) comprising topotecan and cisplatin concurrently. These PEI-GTC-NPs effectively homed into mitochondria in HeLa cervical cancer cells at 6 h and impaired mitochondria leading to reactive oxygen species generation followed by remarkably improved cancer cell death. This platform can be used for specific subcellular organelle targeting for future cancer therapy.

Lipid Nanoparticle-Mediated Induction of Endoplasmic Reticulum Stress in Cancer Cells.

The endoplasmic reticulum (ER) primarily guides protein synthesis, folding, transport, and lipid biosynthesis inside the cells. As a result, dysregulation in those cellular functions leading to ER stress has recently emerged as one of the hallmarks of cancer. Yet, precise navigation in the ER in cancer cells has continued to be a formidable task. Herein, we engineered a lipid nanoparticle (17AAG-ER-NP) containing (a) ER targeting moiety (Tosyl), (b) fluorescent tag with DNA damaging capability (1,8-naphthalimide), and (c) ER stress inducer (17AAG, Hsp90 inhibitor). These lipidic nanoparticles were confined in the ER of HeLa cells over 6 h through caveolin-controlled endocytosis confirmed by confocal microscopy. Western blot analysis, fluorescent microscopy, and flow cytometry studies confirmed that 17AAG-ER-NPs can concurrently activate ER stress and nuclear DNA impairment for arresting the cell cycle in the G2-M phase to elicit late apoptosis, followed by cell death, in a greatly augmented manner compared to free drugs. Interestingly, this nanoparticle-mediated ER stress activated autophagy, which was suppressed through a cocktail treatment with 17AAG-ER-NPs and chloroquine (autophagy inhibitor), prompted remarkable HeLa cell killing at submicromolar concentration. This nanoplatform can support new tools to impair multiple targets in the ER for future cancer therapy.

Polymer conjugated graphene-oxide nanoparticles impair nuclear DNA and Topoisomerase I in cancer.

Cancer chemotherapy had been dominated by the use of small molecule DNA damaging drugs. Eventually, the emergence of DNA damage repair machinery in cancer cells has led to combination therapy with the DNA topology controlling enzyme, topoisomerase I inhibitor along with DNA impairing agents. However, integrating multiple drugs having diverse water solubility and hence bio-distribution effectively for cancer treatment remains a significant challenge, which can be addressed by using suitable nano-scale materials. Herein, we have chemically conjugated graphene oxide (GO) with biocompatible and hydrophilic polymers [polyethylene glycol (PEG) and ethylene-diamine modified poly-isobutylene-maleic anhydride (PMA-ED)], which can encompass highly hydrophobic topoisomerase I inhibitor, SN38. Interestingly, these sheet structured GO-polymer-SN38 composites self-assembled into spherical nanoparticles in water after complexing with a hydrophilic DNA damaging drug, cisplatin. These nanoparticles showed much improved colloidal stability in water compared to their drug-loaded non-polymeric counterpart. These SN38 and cisplatin laden GO-polymer nanoparticles were taken up by HeLa cancer cells through clathrin-dependent endocytosis to home into lysosomes within 6 h, as confirmed by confocal microscopy. A combination of gel electrophoresis, flow cytometry, and fluorescence microscopy showed that these nanoparticles damaged nuclear DNA and induced topoisomerase I inhibition leading to apoptosis and finally improved HeLa cell death. These self-assembled GO-polymer nanoparticles can be used for strategic impairment of multiple cellular targets involving hydrophobic and hydrophilic drugs for effective combination therapy.

Cisplatin-induced self-assembly of graphene oxide sheets into spherical nanoparticles for damaging sub-cellular DNA.

This report describes the hitherto unobserved cisplatin induced self-assembly of 2D-graphene oxide sheets into 3D-spherical nano-scale particles. These nanoparticles can encompass dual DNA damaging drugs simultaneously. A combination of confocal microscopy, gel electrophoresis and flow cytometry studies clearly demonstrated that these novel nanoparticles can internalize into cancer cells by endocytosis, localize into lysosomes, and damage DNA, leading to apoptosis. Cell viability assays indicated that these nanoparticles were more cytotoxic towards cancer cells compared to healthy cells.