Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity

Abstract

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition–fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (Đ ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

Zwitterionic Amino Acid Polymer-Grafted Core-Crosslinked Particle toward Tumor Delivery

“…The chemical structure of the side chain in pGlnMA was the same as that of a reported glutamine-functionalized pLys, which was also internalized into BxPC3 via recognition by ASCT2. The cellular uptake behavior of pGlnMA-CCP in the present study might be similar; however, BzlSer has the potential to bind to other AATs; therefore, more detailed experiments are required to determine if this is an ASCT2-specific interaction.…”

“…A strong affinity of glutaminecontaining polymers for cancer cells expressing ASCT2 has been reported, although the AATs on cancer cells that recognize these polymers are still under discussion. 20,28 ASCT2 is overexpressed on various types of cancer cells, such as CT26 29 and 4T1 cells. 30 For the expression level of on CT26 and 4T1 cells by Western blotting, it was observed that CT26 cells expressed ASCT2 1.2 times as high as 4T1 cells (Figure S14).…”

“…It was observed that the glutamine-functionalized pLys was recognized by the alanine serine cysteine transporter 2 (ASCT2) on human pancreatic cancer cells (BxPC3) and internalized into the cells via the AAT-mediated endocytosis pathway. Methacrylamide- and acrylate-based ZAPs with phenylalanine, lysine, or glutamine in the side chain were also actively internalized into cancer cells via a similar pathway. , Furthermore, the biocompatibility of ZAPs is promising owing to their zwitterionic nature. A previous study demonstrated that at an extremely low level, protein adsorption onto a polymer brush comprising a cysteine-based ZAP was similar to that comprising PEG and phosphobetaine-based zwitterionic polymers .…”

“…Methacrylamide-and acrylate-based ZAPs with phenylalanine, lysine, or glutamine in the side chain were also actively internalized into cancer cells via a similar pathway. 19,20 Furthermore, the biocompatibility of ZAPs is promising owing to their zwitterionic nature. A previous study demonstrated that at an extremely low level, protein adsorption onto a polymer brush comprising a cysteinebased ZAP was similar to that comprising PEG and phosphobetaine-based zwitterionic polymers.…”

Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer–N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines

“…The labeling efficiency was analyzed using UV–vis spectroscopy and calculated by the determination of the molar concentration of dye relative to the molar concentration of polymer (λ max = 646 nm, ε = 230,400 M –1 cm –1 ). All polymers showed a similar incorporation of Cy5, which was determined to be 3–4 dyes per polymer chain (Table ) and which was a particularly critical parameter to use for accurate comparisons in subsequent cell studies . With a focus on different shape/size effect on cell interactions, small-angle neutron scattering (SANS) was further used to analyze the size and shape of the polymeric nanoparticles.…”