Category: Formulation and Quality
Purpose: Breast cancer is most common type of cancer in female with high mortality. Paclitaxel (PTX) is widely used for the treatment of breast cancers. However, its hydrophobic nature with very less water solubility limits its clinical application. Thus, in the present study, anacardic acid (AA) was explored as a hydrophobization moiety for gelatin to increase the payload of gelatin nanoparticles in case of hydrophobic drugs like PTX. Also, AA was used as a functional ligand to achieve the targeted and enhanced therapeutic efficacy of PTX by targeting angiogenesis marker VEGF receptors overexpressed on endothelial cells in tumor environment and cancer cells.
Methods: Gelatin was conjugated with AA using carbodiimide coupling reaction (EDC, NHS) for hydrophobization of gelatin. The AA-modified hydrophobized gelatin was characterized by NMR, FTIR, circular dichroism (CD) spectroscopy and fluorescence spectroscopy. PTX-loaded hydrophobized gelatin nanoparticles were prepared by the nanoprecipitation method. Different formulation and process variables such as % theoretical drug loading, type of stabilizer and homogenization speed were extensively optimized. Lutrol F-87 and poly (acrylic acid, sodium salt) was selected as stabilizer and cross-linking agent respectively. Further, lyophilization was done to enhance the storage stability of nanoparticles. The optimized nanoparticles were evaluated for particle size, polydispersity index (PDI), zeta potential and % entrapment efficiency (% EE). The shape and surface morphology of nanoparticles were determined by SEM. Also, the solid-state properties of nanoparticles were characterized using powder x-ray diffraction (PXRD) and differential scanning calorimetry (DSC). Release study was carried out using dialysis bag method. In vitro haemolysis was checked for the formulation as compared to marketed formulation. Cellular uptake, cell internalization pathway, nuclear colocalization, cytotoxicity and apoptosis study were accomplished in in vitro cell culture studies utilizing MCF-7 cell line. Further, pharmacokinetic and tumor inhibition study was carried out in vivo in Sprague Dawley rats. In vivo toxicity was evaluated in Swiss mice based on AST, ALT, BUN, Creatinine level and effect on RBC morphology in comparison with two marketed formulation Intaxel® and Nanoxel™.
Results: Nanoparticles (Size 275±25nm, PDI 0.3±0.024) with entrapment efficiency of 74% were obtained. The SEM analysis showed the spherical shape of the prepared nanoparticles. PXRD revealed amorphous nature of the nanoparticles after freeze drying and DSC confirmed the entrapment of the drug within the nanoparticles. In vitro release from the dialysis bag was 49% in 24 hours from the nanoparticles as compared to 90 % in 8 hours for free drug. As compared to marketed formulation, nanoformulation showed significantly less hemotoxicity. In vitro cell culture study revealed clathrin- and caveolae-dependent endocytosis of nanoparticles. Further, colocalization study showed strong nuclear colocalization of AA-conjugated nanoparticles. Significant increase in cellular uptake with ~2.26-fold higher apoptosis index and ~5.86-fold reduction in IC50 value compared with PTX in MCF-7 cell line was indicative of higher efficacy of nanoformulation in vitro. Also, ~3.51 and ~1.36-fold increase in area under the curve compared with Intaxel® and Nanoxel™ was achieved. Significant tumor burden reduction (∼60%) was achieved in nanoformulation as compared to marketed formulation. AST, ALT, BUN and Creatinine levels were comparable to control group for the formulation, whilst marketed formulation depicted higher levels of the toxicity markers. Similar pattern of toxicity was also obtained in RBC morphology with SEM imaging.
Conclusion: The nanoformulation was found to be safe and efficacious, with sustained release and higher loading of PTX. The present proof-of-concept experimental data strongly demonstrated that hydrophobization of gelatin with functional ligands like AA offer an additional research horizon to overcome the limitations of hydrophobic drugs like PTX. Thus, the present formulation strategy can be set as a prototype for all difficult-to-deliver hydrophobic drugs showcasing poor solubility and dose-related toxicity with limited clinical efficacy.
Rohan Ghadi– Mohali, Punjab, India
Madhura Rege– Mohali, Punjab, India
Sameer Katiyar– Mohali, Punjab, India
Varun Kushwah– Mohali, Punjab, India
Sanyog Jain– Associate Professor, National Institute for Pharmaceutical Education and Research (NIPER), S.A.S. Nagar, Mohali, Punjab, India