*Article* **Influence of the Topology of Poly(L-Cysteine) on the Self-Assembly, Encapsulation and Release Profile of Doxorubicin on Dual-Responsive Hybrid Polypeptides**

**Dimitra Stavroulaki 1, Iro Kyroglou 1, Dimitrios Skourtis 1, Varvara Athanasiou 1, Pandora Thimi 1, Sosanna Sofianopoulou 2, Diana Kazaryan 1, Panagiota G. Fragouli 3, Andromahi Labrianidou 4, Konstantinos Dimas 4, Georgios Patias 5, David M. Haddleton <sup>5</sup> and Hermis Iatrou 1,\***


**Abstract:** The synthesis of a series of novel hybrid block copolypeptides based on poly(ethylene oxide) (PEO), poly(L-histidine) (PHis) and poly(L-cysteine) (PCys) is presented. The synthesis of the terpolymers was achieved through a ring-opening polymerization (ROP) of the corresponding protected *N*-carboxy anhydrides of *Nim*-*Trityl*-L-histidine and *S*-*tert*-butyl-L-cysteine, using an end-amine-functionalized poly(ethylene oxide) (*m*PEO-NH2) as macroinitiator, followed by the deprotection of the polypeptidic blocks. The topology of PCys was either the middle block, the end block or was randomly distributed along the PHis chain. These amphiphilic hybrid copolypeptides assemble in aqueous media to form micellar structures, comprised of an outer hydrophilic corona of PEO chains, and a pH- and redox-responsive hydrophobic layer based on PHis and PCys. Due to the presence of the thiol groups of PCys, a crosslinking process was achieved further stabilizing the nanoparticles (NPs) formed. Dynamic light scattering (DLS), static light scattering (SLS) and transmission electron microscopy (TEM) were utilized to obtain the structure of the NPs. Moreover, the pH and redox responsiveness in the presence of the reductive tripeptide of glutathione (GSH) was investigated at the empty as well as the loaded NPs. The ability of the synthesized polymers to mimic natural proteins was examined by Circular Dichroism (CD), while the study of zeta potential revealed the "stealth" properties of NPs. The anticancer drug doxorubicin (DOX) was efficiently encapsulated in the hydrophobic core of the nanostructures and released under pH and redox conditions that simulate the healthy and cancer tissue environment. It was found that the topology of PCys significantly altered the structure as well as the release profile of the NPs. Finally, in vitro cytotoxicity assay of the DOX-loaded NPs against three different breast cancer cell lines showed that the nanocarriers exhibited similar or slightly better activity as compared to the free drug, rendering these novel NPs very promising materials for drug delivery applications.

**Keywords:** polypeptides; drug delivery; nanoparticles; doxorubicin; cancer; topology of poly-L-cystein
