Dual affinity nanoparticles for the transport of therapeutics from carrier cells to target cells under physiological flow conditions

Introduction:: High patient circulating tumor cell (CTC) count correlates with poor overall and progression-free survival in most cancer types [1]. Most CTCs are not able to survive the forces and interactions in the circulation, yet few CTCs are needed to successfully form a metastatic site. Therefore, eliminating CTCs before they are able to extravasate and form a metastatic site is an important treatment mechanism that needs to be tackled. In this study, we developed two-stage nanoparticle delivery platform relying on the dual functionalization of a liposome with moieties that have different strengths of adhesion and binding kinetics to attach to a carrier cell (leukocyte) and a target cell (CTC) (Figure 1A). The dual affinity (DA) liposomes have E-selectin and anti-cell surface vimentin (CSV) half antibodies on the surface. Additionally they carry the immunotherapy tumor necrosis factor related apoptosis inducing ligand (TRAIL). The first stage of the delivery mechanism is the nanoparticles tethering to healthy leukocytes through the ES/ESL bond which is weak and reversible [2]. This allows the nanoparticles to be transported and protected by the carrier cell in the circulation. The second stage of the system occurs once the decorated leukocytes collide with the CTC. The liposomes detach from the leukocyte and bind to the vimentin on the CTC surface. Lastly, once the liposome has been transferred from the surface of the leukocyte to that of the cancer cell, the TRAIL can induce apoptosis.


Materials and Methods:: Liposomes were synthesized following the protocol from Mitchell et al [3]. To understand the binding of the DA liposome system to different cell types, four different functionalized liposomes were prepared: bare, anti-CSV half-antibody (CSV), ES, anti-CSV half-antibody + ES (DA). Half antibodies were produced by incubating the unconjugated monoclonal human anti-cell-surface-vimentin antibody with the reducing agent 2-MEA. Liposomes were incubated overnight at 4°C on a rotator to achieve a final concentration of 6 half-antibodies and 2 recombinant human his-tagged ES. The neutrophil-like cancer cell line PLB985 and the colorectal cancer cell line HCT116 were utilized throughout this study. Cone-and-plate viscometers (Brookfield) equipped with a CP-40 spindle were used to simulate the FSS that immune cells experience in the circulation [4]. Binding capacity of the liposomes to both the PLB985 cells was tested by applying uniform FSS to 500,000 cells with 10µL of the liposome solution at 188 s^-1 for 30 min at room temperature. Transfer efficacy of the DA liposomes from the PLB985 cells to the HCT116 cells was tested. PLB985 cells (500,000) were sheared with 10µL of the liposome solution for 30 minutes. Samples were then washed via centrifugation, spiked with 500,000 HCT116 cells, and sheared for 2 hrs. Static controls were performed by repeating the steps above but placing the samples on a rocker instead of the viscometers. Binding of liposomes to PLB985 cells, HCT116 cells, and transfer between cells was confirmed by flow cytometry using the Red-B laser.


Results, Conclusions, and Discussions:: In this study, we prepared liposomes which can adhere reversibly to healthy leukocytes and strongly to CTCs in the circulation. Functionalization was confirmed by dynamic light scattering measurements of the liposome diameter on a Malvern Panalytical Advanced Series Ultra Zetasizer (Figure 1B). The percentage of PLB985 cells decorated with liposomes was quantified using flow cytometry. Under FSS conditions, 36% of the cells in both the ES liposome group and the DA liposome group were decorated (Figure 1C). No significant levels of binding occurred for the bare liposomes and the CSV liposomes compared to the control where no liposomes were present in the sample. When the experiment was repeated with static conditions, the percentage of cells decorated with liposomes decreased significantly by 15-fold and 8-fold in the ES and DA groups, respectively.

Flow cytometry analysis showed an 8-fold increase in the percentage of HCT116 cells decorated with the CSV, ES and DA liposomes compared to the bare liposome group (Figure 1D). Interestingly, the ES liposomes bound to the HCT116 cells to the same degree as the CSV liposomes. Many cancers do express E-selectin ligands, which play a role in extravasation and migration, and survival in the circulation[5]. When the liposomes were incubated with the HCT116 cells under static conditions, there was a significant decrease in binding to the CSV, ES, and DA liposomes. The DA liposomes had a significantly higher transfer efficacy than all other liposomes formulations (Figure 1E). The percentage of HCT116 cells decorated with DA liposomes was 23%, this was a 3-fold increase compared to the percentage of decorated HCT116 cells incubated with the ES liposomes. Interestingly, the ES liposomes did transfer under shear conditions, but had a low efficacy in doing so only transferring to 10% of the HCT116 cell population. As expected, when the transfer efficacy was tested in static conditions, there was a significant decrease in the transfer of the CSV, ES and DA liposomes. We therefore successfully designed and validated a nanoparticle system which can transfer between a carrier cell and a target cell under physiologically relevant FSS conditions found in the circulation.


Acknowledgements (Optional): :

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[2] A. D. Rocheleau, T. M. Cao, T. Takitani, and M. R. King, “Comparison of human and mouse E-selectin binding to Sialyl-Lewisx,” BMC Struct Biol, vol. 16, no. 1, p. 10, Jul. 2016, doi: 10.1186/s12900-016-0060-x.

[3] M. J. Mitchell, E. Wayne, K. Rana, C. B. Schaffer, and M. R. King, “TRAIL-coated leukocytes that kill cancer cells in the circulation,” Proceedings of the National Academy of Sciences, vol. 111, no. 3, pp. 930–935, Jan. 2014, doi: 10.1073/pnas.1316312111.

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