Tuned Density of Anti-Tissue Factor Antibody Fragment onto siRNA-Loaded Polyion Complex Micelles for Optimizing Targetability into Pancreatic Cancer Cells

Abstract

Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azidefunctionalized poly(ethylene glycol) and poly(L-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nano-particle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited similar to 40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.