Formulation and Characterization of Doxycycline loaded Chitosan Nanoparticles for Testing Antitumor Efficacy in Cervical Cancer

Abstract

Cervical cancer is a widespread form of cancer common among women all over the world. It is ranked as the third most common form. Its impact is particularly devastating in Pakistan, leading to the loss of more than 7000 women's lives each year. Chemotherapy and radiotherapy serve as the primary treatment methods for patients diagnosed with metastatic and recurring cervical cancer. While these conventional approaches have shown promise in improving the prognosis of cervical cancer, they often come with significant side effects that can be difficult for patients to endure. Nano oncology, a vital field within nanotechnology, focuses on utilizing nanobiotechnology in cancer treatment. By utilizing nanotechnology, synthesis of chemotherapy DDS in the form of NPs has displayed immense potential in enhancing drug bioavailability, solubility, modifying drug distribution, combating drug resistance, and reducing nonspecific toxicity. The innovative drug delivery system described in this study utilizes a sustained and controlled release mechanism, combined with active targeting through surface-functionalization using a ligand coating. Doxycycline (DOX) as prepared by ionic gelation method, was characterized physiochemically and biologically. In silico studies provided evidence of the strong affinity between hyaluronic acid (HA) and the CD44 receptors, which are highly expressed in Cervical Cancer. This finding was further supported by GEPIA analysis, which confirmed high CD44 expression in Cervical Squamous Cell Carcinoma, as well as in HeLa cells according to the Human Atlas. Additionally, STRING analysis displayed that CD44 has a significant role in metastasis and progression of cancer. The mitochondrial biogenesis inhibitor Doxycycline NFs formulated demonstrated encapsulation efficiency of 89%. In Vitro release assays validated the controlled release pattern of drug within a 72- hour period in an acidic environment similar to tumor microenvironment. The anticancer activity of raw DOX at doses of 20 to 100 µg/ml on HeLa cervical cancer cells reported IC50 of 26 µg/ml in 2 comparison to DOX NFs that exhibited an IC50 value of 13 µg/ml on the same cells. This confirmed the strong cytotoxic potential of NFs by an MTT assay. RT-PCR analysis showed downregulation of TNF-Alpha in response to doxycycline. Overall, these findings highlight the potential of this novel drug delivery system in targeted treatment for Cervical Cancer, offering sustained release of Doxycycline while minimizing drug toxicity and improving therapeutic outcomes.