(M0930-06-36) Development and Evaluation of Ribociclib-Loaded Folic Acid Conjugated Chitosan-Polycaprolactone Nanoparticles for the Management of Glioblastoma

Purpose: Glioblastoma (GBM) is a complex and aggressive type of brain cancer, with a five-year survival rate of only 6.9%. In 2024, approximately 14,000 people were diagnosed with glioblastoma in the United States. Chemotherapy, surgical resection, and radiation therapy are the major current treatment options for Glioblastoma. Temozolomide, Bevacizumab, Carmustine, and Lomustine are widely used chemotherapeutic drugs for Glioblastoma. Ribociclib, a CDK 4/6 inhibitor, is approved for the treatment of hormone receptor (HR) positive and HER2 negative breast cancer. Ribociclib has been reported to cross the blood-brain barrier (BBB), showing promising effects in treating glioblastoma. Over the years, the anti-cancer agents have suffered from off-target toxicity and non-specific effects in preclinical and clinical setups. Active targeting of specific cancer cells by targeting the overexpressed receptors through conjugating the carrier system with a suitable ligand is a possible approach to overcome this burden. Folate receptors are widely overexpressed in many tumors, including glioblastoma. These membrane-bound surface proteins can bind to folate and folate conjugates with higher affinity. We aim to fabricate and optimize the folic acid conjugated chitosan-polycaprolactone nanoparticle and extensively characterize and investigate the anti-cancer efficacy of Ribociclib and Ribociclib-loaded formulations in 2-dimensional and 3D spheroid cultures of GBM cell lines.

Methods: Folic acid (FA) was conjugated to chitosan (CS) by carbodiimide chemistry, and the formation of the amide bond between the FA and CS was confirmed by ¹H NMR and ATR-FTIR spectroscopic techniques. The ionic gelation method was used to fabricate the Ribociclib-loaded folic acid conjugated chitosan-polycaprolactone nanoparticles (RNPs). Briefly, the weighed quantity of FA conjugated chitosan (FCS) was dissolved in the aqueous phase. Simultaneously, the weighed quantity of Ribociclib and polycaprolactone was dissolved in a homogenous mixture of acetone and methanol (organic phase). Then, the drug-containing organic phase was slowly added to the aqueous phase under stirring and kept for overnight stirring for complete removal of organic solvents. The RNPs were extensively evaluated by different physicochemical characterization techniques, including ¹H NMR, ATR-FTIR, DSC, particle size, PDI, zeta potential, morphology, % entrapment efficiency, drug loading, and in-vitro drug release studies. The anti-cancer efficacy of optimized RNPs was assessed in glioblastoma cells by multiple assays, such as in-vitro cytotoxicity, receptor blocking assay, qualitative and quantitative cellular uptake assays, reactive oxygen species assay, qualitative apoptosis assay (acridine orange and ethidium bromide dual staining), mitochondrial membrane potential assay, in-vitro cellular migration, and clonogenic assay. Cell viability, spheroid volume, and live/dead assay in Spheroid cultures of U87 MG cells were used to study the efficacy of RNPs.

Results: An analytical method for Ribociclib was developed at 276 nm using ultra-performance liquid chromatography, and the R² value of the calibration curve was found to be 0.9959. The conjugation between the FA and CS was confirmed by a broad singlet peak at 8.35 ppm in ¹H NMR spectra suggesting the formation of amide bond and peaks at 1630 cm^{− 1}, 1560 cm^{− 1} in ATR-FTIR spectra corresponding to the stretching of bonded C=O group and N–H bending vibration of the secondary amide group. RNPs were fabricated by the single-step emulsification method, and the particle size, PDI, and zeta potential of the optimized RNPs were found to be 118.2 ± 9.5 nm, 0.175 ± 0.009, and 5.5±1.2 mV, respectively, as depicted in Table 1. The optimized RNPs exhibited a % drug entrapment of 70.8±4.3%, equivalent to 3.4±0.3% drug loading. Nearly 100% of encapsulated Ribociclib was released from RNPs in pH 5.5 phosphate buffer, whereas 97% of Ribociclib was released from RNPs in pH 6.4 simulated nasal fluid. The RNPs exhibited approximately 8.33 and 10.4-fold reduction in IC₅₀ values compared to pure Ribociclib in U-87 MG and U-138 MG cells for 48 h, respectively, as shown in Figure 1. RNPs exhibited extensive mitochondrial depolarization, enhanced ROS generation, apoptosis (acridine orange/ ethidium bromide dual staining) (Shown in Figure 2), reduced % cell migration, and colony formation compared to pure Ribociclib.

Conclusion: In this study, we fabricated and evaluated Ribociclib-loaded folic acid conjugated chitosan-polycaprolactone nanoparticles. RNPs exhibited enhanced anti-cancer efficacy of Ribociclib in monolayer and spheroid culture of glioblastoma cells. Thus, it was reported that delivering Ribociclib with folic acid-conjugated chitosan-polycaprolactone nanoparticles presents an optimistic approach to treating glioblastoma.


Table 1. Optimization of RNPs


Fig. 1 In-vitro cytotoxicity studies of Ribociclib and RNPs in a) U87b MG and b) U138 MG cells


Fig. 2 Qualitative cellular studies of Ribociclib and RNPs in U87 MG cells a) DCFDA assay b) Acridine Orange / Ethidium Bromide apoptosis assay and c) Mitochondrial Membrane potential