ISIS-5132

A novel gene transfection vector was fabricated based on the conjugation of human serum albumin (HSA) and maleimide end functionalized poly[(N,N‐dimethylamino) ethyl methacrylate] (PDMAEMA). The bioconjugation was achieved in a site‐specific manner to yield well‐defined polymer‐protein conjugates. The biohybrid was able to bind DNA with high affinity resulting in nanoparticles with a HSA shell. This paper mainly focuses on the influence of polymeric chain length on the particle properties and their drug‐carrying ability to deliver oligonucleotides into breast cancer cells. The cytotoxic agent of interest, ISIS5132, is an oligonucleotide which disrupts DNA function within the cell. There was no evidence that the polymeric chain length had any effects on the conjugation efficiency and the subsequent condensation ability of the conjugates to oligonucleotide. However, the polymeric chain length had an obvious effect on the size of the complex micelles. Low molecular weights only led to loosely compacted complexes with the oligonucleotide, while large molecular weight led to well‐defined nanoparticle structures. More importantly, it was found that the variation in the length of the PDMAEMA block resulted in a change in cytotoxicity of the drug loaded complex micelle. That is, the concentration of 50% inhibition (IC50) of the complex micelle on MDA‐MB‐231 and MCF‐7 cells reached the lowest value at a chain length of around 21 000 g mol−1. The IC50 value increased when the polymeric chain length was shorter (8000 g mol−1 and 10 000 g mol−1) while it increased again when PDMAMEA of  = 47 000 g mol−1, probably due to insufficient release of the drug. These result were reflected when investigating the performance of the polyplex using MCF‐7 multicellular tumor spheroids, where again the medium PDMAEMA chain length led to the best delivery vehicle for ISIS5132.

Novel biocompatible polyion complex micelles, containing bovine serum albumin (BSA), polymer, and oligonucleotide, were synthesized as a generation of vectors for the gene transfection. Maleimide-terminated poly((N,N-dimethyl amino) ethyl methacrylate) (PDMAEMA) was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and subsequently deprotected. Precise one to one albumin-PDMAEMA bioconjugates have been achieved via 1,4-addition with the free thiol group on Cys34 on the BSA protein. SDS-PAGE and GPC (water) confirmed and quantified the successful conjugation. The conjugation efficiency was found to be independent of the molecular weight of PDMAEMA. After careful pH adjustment, the conjugate could efficiently condense anticancer oligonucleotide, ISIS 5132, which resulted in particles of 15-35 nm with a negative zeta-potential. The size was easily controlled by the polymer chain length. The albumin corona provides complete protection of the cationic polymer and genetic drug, which gave rise to lower potential toxicity from the polymer and higher gene transfection efficiency. Although a control experiment with a traditional PEG-based polyion complex micelle could deliver the drug just as effectively, if not more so, to the ovarian cancer cell line OVCAR-3, this carrier had no selectivity toward cancerous cells and proved just as toxic to HS27 (fibroblast) cell line. In contrast, the albumin-coated particles demonstrated desirable selectivity toward cancerous cells and have been shown to have outstanding performance in the cytotoxicity tests of several carcinoma monolayer cell models. In addition, the complex micelles were able to destroy pancreatic multicellular tumor spheroids, while free ISIS 5132 could not penetrate the spheroid at all. Hence, albumin-coated/oligonucleotide complex micelles are far more promising than the most classical gene delivery vectors.