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Shuyang Sun,1,2 Ziqiang Ding,1 Xiaomei Yang,1,3 Xinyue Zhao,1,3 Minlong Zhao,1,2 Li Gao,1,3 Qu Chen,1,2 Shenxia Xie,1,4 Aiqun Liu,1 Shihua Yin,1 Zhiping Xu,5 Xiaoling Lu1,2 1International Nanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 2School of Stomatology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 4Department of Pharmacology, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of China; 5Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaCorrespondence: Xiaoling LuNanobody Research Center, Guangxi Medical University, Nanning, Guangxi, 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnZhiping XuAustralian Institute for Bioengineering and Nanotechnology, University of Queensland, St Lucia, QLD, 4072, AustraliaEmail gordonxu@uq.edu.auAbstract: The development of monoclonal antibody treatments for successful tumor-targeted therapies took several decades. However, the efficacy of antibody-based therapy is still confined and desperately needs further improvement. Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size (∼ 15kDa), excellent solubility, superior stability, ease of manufacture, quick clearance from blood, and deep tissue penetration, which gain increasing acceptance as therapeutical tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. Thus, one of the promising novel developments that may address the deficiency of monoclonal antibody-based therapies is the utilization of nanobodies. This article provides readers the significant factors that the structural and biochemical properties of nanobodies and the research progress on nanobodies in the fields of tumor treatment, as well as their application prospect.Keywords: nanobody, drug delivery, immunotherapy, CAR-T, tumor treatment

Ziqiang Ding,1,2,* Dujin Wang,1,2,* Wei Shi,2,3,* Xiaomei Yang,2,3 Siliang Duan,2 Fengzhen Mo,2 Xiaoqiong Hou,2,3 Aiqun Liu,2 Xiaoling Lu2,4 1National Center for International Research of Biological Targeting Diagnosis and Therapy, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 2International Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 3School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; 4College of Stomatology, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China*These authors contributed equally to this workCorrespondence: Xiaoling LuInternational Nanobody Research Center of Guangxi, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of ChinaTel/Fax +86 771-2387 518Email luxiaoling@gxmu.edu.cnPurpose: Cancer tissue-specific and nuclei-targeted drug delivery is ideal for the delivery of chemotherapy. However, it has only been achieved in in vitro studies mainly due to low efficiency in vivo. In this study, we aimed to establish an efficient dual-targeted system that targets liver cancer tissue as well as the nuclei of cancer cells in vivo.Methods: We first synthesized TAT peptide (TATp)-mesoporous silica nanoparticle (MSN) complex (TATp-MSN) and generated liposomes that carried liver cancer-specific aptamer TLS11a (TLS11a-LB). We then generated the drug TLS11a-LB@TATp-MSN/doxorubicin (DOX) by mixing TLS11a-LB and DOX-loaded TATp-MSN. After physical and chemical characterization of the nanoparticles, DOX release from these formulations was evaluated at pH 5.0 and 7.4. Furthermore, we also evaluated nuclear localization and cytotoxicity of the drug in H22 cells in vitro and investigated the liver cancer targeting and antitumor activities of the nano-drug in vivo using a H22 tumor-bearing mice model.Results: TLS11a-LB@TATp-MSN/DOX and its controls were confirmed as nano-drugs (< 100 nm) using transmission electron microscopy (TEM). The DOX release rate of TLS11a-LB@TATp-MSN/DOX was significantly faster at pH 5.0 than at pH 7.4. TLS11a-LB@TATp-MSN/DOX effectively targeted the nuclei of H22 cells and released DOX with a higher efficiency than that of the control groups. In addition, TLS11a-LB@TATp-MSN/DOX exhibited slight cytotoxicity, but not significantly more than controls. In vivo studies showed that TLS11a-LB@TATp-MSN accumulated in subcutaneous H22 tumors in the right axilla of BALB/c mice, reaching peak levels at 48 h after intravenous injection, respectively, and demonstrated that TLS11a-LB@TATp-MSN/DOX group enhanced tumor treatment efficacy while reducing systemic side effects.Conclusion: TLS11a-LB@TATp-MSN/DOX can efficiently deliver DOX to the nuclei of liver cancer cells by dual targeting liver cancer tissue and the nuclei of the cancer cells in mice. Thus, it is a promising nano-drug for the treatment of liver cancer.Keywords: targeted drug delivery, liver cancer treatment, MSN-based vehicles, doxorubicin, tissue- and nuclei-specific targeting