Apoptosis-promoting effect of rituximab-conjugated magnetic nanoprobes on malignant lymphoma cells with CD20 overexpression

Lina Song,1,2 Wei Zhang,3 Hong Chen,4 Xizhi Zhang,1 Haoan Wu,1 Ming Ma,1 Zhongqiu Wang,2 Ning Gu,1 Yu Zhang1 1State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering & Collaborative Innovation Centre of Suzhou Nano Science and Technology, Southeast University, Nanjing 210096, People’s Republic of China; 2Department of Radiology, Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, People’s Republic of China; 3The Jiangsu Province Research Institute for Clinical Medicine, The First Affiliated Hospital with Nanjing Medical University, Nanjing 210009, People’s Republic of China; 4Department of Gastroenterology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, People’s Republic of China Background: Cancer targeting nanoprobes with precisely designed physicochemical properties may show enhanced pharmacological targeting and therapeutic efficacy. As a widely used commercialized antibody, rituximab has been in clinical use for three decades and has lengthened or even saved thousands of lives. However, many people cannot benefit from rituximab treatment because of drug resistance or side effects.Methods: In this study, a 13-nm rituximab-conjugated magnetic nanoparticle was developed as a therapeutic nanoprobe targeting CD20 overexpressing malignant lymphoma cells to enhance the treatment effects of rituximab. The magnetic cores (2,3-dimercaptosuccinicacid modified Fe3O4 nanoparticles, Fe3O4@DMSA) of the nanoprobes with an average diameter of 6.5 nm were synthesized using a co-precipitation method. Rituximab was then conjugated on the surface of Fe3O4@DMSA using a cross-linking agent (carbodiimide/N-hydroxysulfosuccinimide sodium salt). Based on theoretical calculations, approximately one antibody was coupled with one nanoparticle, excluding the multivalent antibody effect.Results: Cell targeting experiments and magnetic resonance (MR) signal and T2 measurements showed that the Fe3O4@DMSA@Ab nanoprobes have specific binding affinity for CD20-positive cells. Compared to rituximab and Fe3O4@DMSA, Fe3O4@DMSA@Ab nanoprobes significantly reduced cell viability and promoted Raji cell apoptosis. Initiating events of apoptosis, including increased intracellular calcium and reactive oxygen species, were observed in nanoprobe-treated Raji cells. Nanoprobe-treated Raji cells also showed the most drastic decrease in mitochondrial membrane potential and Bcl-2 expression, compared to rituximab and Fe3O4@DMSA-treated Raji cells.Conclusion: These results indicate that Fe3O4@DMSA@Ab nanoprobes have the potential to serve as MRI tracers and therapeutic agents for CD20-positive cells. Keywords: CD20-targeted nanoprobes, calcium, reactive oxygen species, ROS, mitochondrial membrane potential, MMP, cell apoptosis &nbsp