Protein delivery nanosystem of six-arm copolymer poly(ε-caprolactone)–poly(ethylene glycol) for long-term sustained release

Jianwei Duan,1,* Chao Liu,1,* Xiaoyu Liang,1 Xuanling Li,1 Youlu Chen,1 Zuoguan Chen,2 Xiaoli Wang,1 Deling Kong,1,3 Yongjun Li,2 Jing Yang1 1Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China; 2Department of Vascular Surgery, Beijing Hospital, National Center of Gerontology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China; 3Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin, China *These authors contributed equally to this work Background: To address the issue of delivery of proteins, a six-arm copolymer, six-arm poly(ε-caprolactone)–poly(ethylene glycol) (6S-PCL-PEG), was synthesized by a simple two-step reaction. Thereafter, the application of 6S-PCL-PEG as a protein carrier was evaluated. Materials and methods: A six-arm copolymer, six-arm poly(ε-caprolactone) (6S-PCL), was synthesized by ring-opening polymerization, with stannous octoate as a catalyst and inositol as an initiator. Then, poly(ethylene glycol) (PEG) was linked with 6S-PCL by oxalyl chloride to obtain 6S-PCL-PEG. Hydrogen-1 nuclear magnetic resonance spectrum, Fourier-transform infrared spectroscopy, and gel-permeation chromatography were conducted to identify the structure of 6S-PCL-PEG. The biocompatibility of the 6S-PCL-PEG was evaluated by a cell counting kit-8 assay. Polymeric nanoparticles (NPs) were prepared by a water-in-oil-in-water double emulsion (W1/O/W2) solvent evaporation method. The size distribution and zeta potential of NPs were determined by dynamic light scattering. Transmission electron microscopy was used to observe the morphology of NPs. Drug-loading capacity, encapsulation efficiency, and the release behavior of ovalbumin (OVA)-loading NPs were tested by the bicinchoninic acid assay kit. The stability and activity of OVA released from NPs were detected and the uptake of NPs was evaluated by NIH-3T3 cells. Results: All results indicated the successful synthesis of amphiphilic copolymer 6S-PCL-PEG, which possessed excellent biocompatibility and could formulate NPs easily. High drug-loading capacity and encapsulation efficiency of protein NPs were observed. In vitro, OVA was released slowly and the bioactivity of OVA was maintained for over 28 days. Conclusion: 6S-PCL-PEG NPs prepared in this study show promising potential for use as a protein carrier. Keywords: six-arm PCL-PEG, copolymer synthesis, protein carrier, sustained releas