Green synthesis of oncolytic Newcastle disease virus-loaded thiolated chitosan nanoformulation for CD44 targeted delivery and sustained release of virus in cervical cancer xenografts

Abstract

BackgroundNewcastle disease virus (NDV) Lasota strain has proven oncolytic activity. One shortcoming associated with this treatment modality is the immune neutralization of the virus in body, mediated by natural killer cells and macrophages. This study aims at encapsulating the oncolytic virus in thiolated chitosan nanoparticles, surface functionalized with hyaluronic acid for CD44 targeted delivery and sustained release of NDV in cervical cancer tumors. These nanoparticles were tested for oncolytic potential in HeLa cells and cervical cancer xenograft model.ResultsNDV-loaded nanoparticles were prepared using TCs concentration of 1.0 mg/mL, HA at 0.5 mg/mL, with a half dose (not less than 500 TCID units) of NDV by using green synthesis approach through ionic gelation method. Viral quantification in nanoparticles was done by TCID50 (50% tissue culture infectious dose) and MOI (multiplicity of infection) determination. Ex vivo NK cell activity was analyzed by quantifying levels of IFN-γ. In vivo analysis was performed on wistar rats, immunocompromised by using ketoconazole (10 mg/kg) and cyclosporin (30 mg/kg) along with 0.1 μg/mL of amoxicillin. WBC profiling on day 7 confirmed immunosuppression, which was followed by tumor implantation. Zeta analysis of NDV nanoparticles showed that nanoparticles are 286.9 nm in size with a zeta potential of 18.1 mV and PDI of 0.241. For estimation of anticancer potential, MTT and trypan blue exclusion assay revealed a higher cytotoxic potential of the encapsulated virus, while TCID50 of HA-TC-NDV was 4.1 as compared to naked NDV virus which had TCID50 of 6.0 on HeLa cells. Histopathology of organs from NDV nanoparticle-treated rats showed syncytia formation in tumors, immunohistochemical analysis showed decrease in expression of TNF-α, COX-II and NF-ƙB which was also confirmed by ELISA. RT-PCR showed high viral copy number in tumor tissue and viral accumulation in lungs and liver. Lower IFN-γ in nanoparticles treated rats showed suppression in immunoreactivity of virus-loaded nanoparticles.ConclusionOur findings suggest that encapsulation of the virus in thiolated chitosan and CD44 targeting enhanced retention and sustained release of the virus in tumors as compared to pure NDV, with increased oncolytic effect both in vitro and in vivo.