Delivery of chemotherapeutic drug targeting folate receptor to oral cancer cells using functionalized carbon nanospheres.

Folate receptor (FR) (α) has long been the subject of active interest as regards its potential to serve as a target for cancer therapy. FR has been found to be overexpressed in several cancers, including clinical samples of different stages from OSCC (oral squamous cell carcinoma) patients. However, no clear correlation or conclusive finding has been obtained so far which might indicate the efficacy of FR as a credible target for the treatment of OSCC. All cell lines to be used were assessed for FR-expression. Subsequently, we developed glucose-derived carbon nanospheres (CSPs) and primed them with a Folate-based cationic lipid FA8 and the chemotherapeutic drug doxorubicin (DOX). CSP based delivery systems along with pristine drug DOX were characterized and treated subsequently toin vitrocultures of OSCC cells and assessed for cancer cell targetability as well as cell death. Subsequently, treatment was administered to immunocompetent C57 mice carrying MOC2 based syngeneic OSCC tumours and assessed for tumour regression and toxicity. Ligand primed targeted CSPs exhibited commendable drug uptake as well as efficient induction of cell death. Further, receptor blocking studies revealed FR-mediated uptake, preferentially in cancer cells. Drug once delivered by ligand-primed CSPs was retained longer inside cells than pristine drug alone, indicating possibilities of better therapeutic outcome. In animal studies, CSP-FA8-DOX (Ligand primed targeted CSP) demonstrated significant regression in tumour size compared to pristine DOX as well as CSP-DOX (non-targeted CSP) treated animals. FR-mediated system CFD demonstrated targeted drug uptake and apoptotic death selectively in cancer cells. Significant tumour regression was also observedin vivo. Overall, it may be presumed that the FR is a therapeutic target with substantial potential in OSCC treatment.

Allosteric Regulation of Cyclin-B Binding by the Charge State of Catalytic Lysine in CDK1 Is Essential for Cell-Cycle Progression.

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.

Dual targeting of folate receptor-expressing glioma tumor-associated macrophages and epithelial cells in the brain using a carbon nanosphere-cationic folate nanoconjugate.

Glioblastoma multiforme (GBM), the highly invasive form of glioma, exhibits the highest mortality in patients with brain malignancies. Increasing glioma patients' survivability is challenging, as targeting only tumor-associated malignant cells would not reduce the overall aggressiveness of the tumor mass. This is due to the inadequacy in countering pro-proliferative, invasive and metastatic factors released by tumor-mass associated macrophages (TAMs). Hence, strategically, dual targeting both tumor cells and TAMs is necessary for effective glioma treatment and increased survivability. Conventional FR-targeting systems can easily target cancer cells that overtly express folate receptors (FRs). However, FRs are expressed only moderately in both glioma cells and in TAMs. Hence, it is more challenging to coordinate dual targeting of glioma cells and TAMs with lower levels of FR expression. A recently developed carbon nanosphere (CSP) with effective blood-brain barrier (BBB) penetrability was modified with a new folic acid-cationic lipid conjugate (F8) as a targeting ligand. The uniqueness of the cationic lipid-folate conjugate is that it stably associates with the negatively charged CSP surface at about >22 mol% surface concentration, a concentration at least 5-fold higher than what is achieved for conventional FR-targeting delivery systems. This enabled dual uptake of the CSP on TAMs and tumor cells via FRs. A doxorubicin-associated FR-targeting formulation (CFD), in an orthotopic glioma model and in a glioma subcutaneous model, induced the maximum anticancer effect with enhanced average mice survivability twice that of untreated mice and without any systemic liver toxicity. Additionally, we observed a significant decrease of TAM-released pro-aggressive factors, TGF-ß, STAT3, invasion and migration related sICAM-1, and other cytokines indicating anti-TAM activity of the CFD. Taken together, we principally devised, to the best of our knowledge, the first FR-targeting nano-delivery system for targeting brain-associated TAMs and tumor cells as an efficient glioma therapeutic.