br Results F and F were screened out
Results: F18 and F19 were screened out as HELT fractions in vivo and F18-loaded emulsions of diﬀerent con-centrations for lymphatic administration were prepared. We confirmed that HD-E and MD-E produced obvious antitumor activities in footpad tumors and LNM compared with other groups in vitro. We also verified the eﬀects of F18-loaded emulsions on activating hematopoietic function, stimulating proliferation of the spleen and nat-ural killer (NK) cells, and promoting the secretion of IFN-γ and IgG1, although HD-E performed mild toxicity on liver.
Conclusion: The present study demonstrated that lymphatic chemotherapy with HELT fraction of bufadienolides could be an eﬀective approach to the treatment of CRC patients with LNM.
Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death according to GLOBOCAN 2018 . Lymph node metastasis (LNM) is one of the determinant prognostic factors of CRC . Unfortunately, conventional intravenous che-motherapeutics are diﬃcult to highly concentrate in regional lymph
nodes on account of the anatomical properties of lymphatic system, which results in limited therapeutic eﬀects. Furthermore, the che-motherapeutics rapidly accumulating in normal organs and tissues could inevitably cause severe side eﬀects . Therefore, lymphatic chemotherapy may solve these problems by distributing more drugs in SCR7 and less in blood circulation.
Currently, lymphatic chemotherapy has been proved to have
Correspondence to: Y. Xu, Institute of General Surgery, Chinese General Hospital of People's Liberation Army, No. 28 Fuxing Road, Beijing 100853, PR China.
Correspondence to: X. Du, Department of General Surgery, Chinese General Hospital of People's Liberation Army, No. 28 Fuxing Road, Beijing 100853, PR China. E-mail addresses: [email protected] (Y. Xu), [email protected] (X. Du). 1 These authors contributed equally to this work.
satisfactory antitumor eﬀects on various cancers [4–8]. Previous studies showed that using several drug-loaded carriers [8–12] to absorb or encapsulate chemotherapeutics provided a better penetration into lymphatics. However, the lymphatic uptake rate is not high and the biocompatibility of some carrier materials is not ideal for the lymphatic system, which may lead to some adverse reactions . Currently, drugs rather than drug carriers that being appropriate to lymphatic chemotherapy are needed. At this point, traditional Chinese medicine (TCM) possesses obvious advantages . Bufadienolides, the major active components of the skin of Bufo bufo gargarizans Cantor (toad skin) have shown high antitumor activities and also a broad spectrum to various cancer cell lines, such as human astrocytoma, lung and gastric tumors . However, they also performed high toxicity or serious side eﬀects clinically . Fractions of bufadienolides such as resibufogenin have the antitumor eﬀect as well as toxicity and other side eﬀects [17,18]. Thus, it is essential to identify the fractions of bufadienolides with high antitumor activity and low toxicity for application in lym-phatic chemotherapy, in order to decrease cytotoxicity to the greatest extent.
In the present study, the eﬀects of 22 fractions of bufadienolides on tumor cells and cytokine-induced killer (CIK) cells were evaluated and two fractions (F18 and F19) with high eﬃciency and low toxicity (HELT) were screened. The anticancer eﬃcacy, the eﬀects on immune function, and the potential toxicity of F18 in lymphatic chemotherapy were assessed in a CRC model with lymphatic metastasis.
2. Materials and methods
2.1. Preparation of bufadienolides and standard solution
Bufadienolides separated and purified from the skin of Bufo bufo gargarizans Cantor (toad skin) have been previously described in detail . Briefly, 4 kg dried toad skin was decocted with 95% ethanol. The combined decoctions were dried with rotary evaporation at 60 °C in vacuum. 140 g extract was dissolved in 70% methanol again, and then extracted with n-heptane. Methanol fraction was dried and 40 g residue was dissolved in methanol and filtered through 0.45 μm membrane filter to form the sample of 440 mg/mL. After that, the crude sample was fractionated with preparative high-performance liquid chromato-graphy (prep-HPLC) and 22 fractions (labeled as F1–F22) were col-lected according to the absorption peaks. Finally, the fractions were purified on the water purification factory. Dr. Xiuli Zhang and Prof. Xinmiao Liang from Dalian Institute of Chemical Physics, China Academy of Science (Dalian, China) provided these 22 fractions for further study about pharmacological activities and toxicity in our la-boratory. All fractions were stored at −20 °C.