Associate Professor University of Delaware, United States
Introduction: Breast cancer tumor cells drain from primary tumor sites into the lymph nodes (LNs) and create metastases that significantly worsen survival outcomes for cancer patients (1). Drug transport into the LN from the systemic circulation is largely excluded by a network of high endothelial venules (HEVs) (2), therefore standard chemotherapeutic treatment efficacy of metastatic lesions is greatly reduced. There is a pressing need to develop novel LN-targeted drug carriers to deliver therapeutic doses of chemotherapeutics to the LN. Systemically circulating lymphocytes routinely cross the HEVs and enter the LN (3) where metastasis occurs (4). We have leveraged this ability of lymphocytes to enter the LN to deliver small molecule drugs to the LN. To that end, we have developed cyroshocked T lymphocytes (CSTLs) loaded with the anti-cancer drug doxorubicin (DOX). DOX can be loaded passively via diffusion or actively via fusogenic liposomes. Using both techniques, we can increase the loading efficiency of the CSTLs by ~4 fold. Furthermore, combing these techniques enables compartmentalization of drug loading inside a CSTL for differential temporal release of the drug in each compartment.
Materials and
Methods: Jurkat cells were suspended in a DOX solution and flash frozen in liquid nitrogen to obtain nuclear DOX loaded CSTLs, (DOX-CSTLs). DOX loaded fusogenic liposomes (DOX-FusLipos) or Cy5 loaded fusogentic liposomes (Cy5-FusLipos) were prepared separately by extrusion. FusLipos were incubated with DOX-CSTLs to increase loading efficiency of drug and create dual loaded CSTLs with drug loaded in the cytoplasmic space (Figure 1A). To calculate the loading efficiency of CSTLs, they were dissociated using 1% TritonTM X-100 and the concentration of the released DOX was measured using UV-Vis spectroscopy. Confocal microscopy was used to visualize the localization of DOX inside CSTLs. The total lipid content of the fusogenic liposome treated CSTLs was characterized using enzymatic phospholipid quantification assay and compared to that of untreated controls. To evaluate the in vitro anti-cancer efficacy of DOX-CSTLs, 4T1 breast cancer cells were cultured in a 12-well plate and the apical chamber of a transwell insert was loaded with patched DOX-CSTLs (Figure 1F). 4T1 cell viability was tracked using Alamar blue assay.
Results, Conclusions, and Discussions: Results & Discussions: We developed a novel method to prepare DOX loaded cryo-shocked lymphocytes using fusogenic liposomes to enhance their drug loading efficiency. CSTLs are functionally dead but remain as structurally intact lymphocytes. Our data shows that CSTLs enter the LN through the systemic circulation. However, the amount of drug that can be loaded into the CSTLs via passive loading is relatively low. We sought to create a method to more efficiently load the CSTLs; this was accomplished by actively loading CSTLs with drug-loaded fusogenic liposomes. Post incubation with fusogenic liposomes, membrane surface markers, such as L-selectin, remained on CSTLs and the lipid content of the cell membrane was replenished as confirmed via flow cytometry, and lipid quantitation (Figure 1C) respectively. Consequently, incubation of DOX-CTLSs with DOX-FusLipos allows ~4-fold more DOX to be loaded into the CSTLs as compared to passive or active only loaded CSTLs (Figure 1D). The dual loading strategy also allowed for selectively loading drug into the cytoplasmic or nuclear compartment of the CSTLs. Direct imaging confirmed passively loaded DOX localized in the nucleus, while actively loaded Cy5 localized to the cytoplasm (Figure 1E). Our dual loading method preserves the ability for CSTLs to carry drug into the lymph node by preserving membrane surface receptors, but increases the drug loading per vehicle. Moreover we demonstrated the release and pharmacologic function of loaded DOX in vitro using DOX-CSTL treatment of 4T1 cancer cells (Figure 1F,G). DOX released from dual-loaded CSTLs retained its cytotoxic efficacy for up to 72 h after delivery. Taken together, the high loading efficiency and extended anti-cancer efficacy of DOX-CSTLs indicate they are a promising carrier system targeted to the LN.
Conclusions: We developed a fusogenic liposome-based strategy to improve DOX loading of a lymphocyte derived cryo-attenuated carrier. We have also demonstrated this strategy can not only improve DOX loading efficiency of the CSTLs but also can spatially load different compartments of the CSTLs depending on lipid identity, enabling more advanced pharmacological release kinetics.
