Design of in situ porcine closed-circuit system for assessing blood-contacting biomaterials.

The overall pre-clinical process of determining the blood compatibility of any medical device involves several stages. Although the primary purpose is to protect the patients, laboratory testing has been over-utilized for many years with a huge number of unnecessary animal tests being done routinely. Recently, the elimination of needless testing has become important in controlling the cost of healthcare and in addressing many issues related to the ethics of animal research. With this in mind, we designed a new in situ porcine closed-circuit system to study the complex interplay between platelets, coagulation proteins, and other cellular elements in pigs. We proved that this system can be implemented in blood compatibility testing and minimize the number of animals used in the experiments.

Anti-platelet adhesion and in situ capture of circulating endothelial progenitor cells on ePTFE surface modified with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and hemocompatible peptide 1 (HCP-1).

We recently reported in vitro suppression of platelet adhesion on expanded polytetrafluoroethylene (ePTFE) by surface grafting of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). However, this may be inadequate for long-term hemocompatibility of blood-contacting biomaterials, and it has led us to develop a strategy of circulating mononuclear cell-capture. ePTFE was treated with argon (Ar) plasma, and grafted with 2-methacryloyloxyethyl phosphorylcholine (MPC) and methacrylic acid (MAA), by glycidyl methacrylate (GMA)-anchored graft polymerization. Next, it was immobilized with integrin α4ß1-positive circulating blood cell-specific peptides, i.e., the traditional arginine-glutamic acid-aspartic acid-valine (REDV), and our original hemocompatible peptide-1 (HCP-1). Both the surfaces retained the anti-platelet property just like the PMPC-grafted surface, and revealed considerable affinity to human umbilical vein endothelial cells (HUVEC), which is a well-known in vitro integrin α4ß1-positive model. Better HUVEC spreading and proliferation was also confirmed, in terms of the cell extension property. Since coagulation and endothelialization on the materials compete in the body, they cannot be properly evaluated separately, in vitro. They were assessed by using an in situ porcine closed-circuit system for 18 h in the present study. Our findings suggest that poly(MPC-co-MAA) is a great ePTFE surface modifier, exhibiting good hemocompatibility in association with REDV/HCP-1 immobilization, which suppresses anti-platelet adhesion and enhances circulating cell capture simultaneously.

A surface graft polymerization process on chemically stable medical ePTFE for suppressing platelet adhesion and activation.

An effective surface grafting method for chemically inert and elaborately porous medical expanded-polytetrafluoroethylene (ePTFE) was developed. Although surface graft polymerization onto basic polymeric biomaterials has been widely studied, successful modification of the ePTFE surface has been lacking due to its high chemical resistance. Herein, we succeeded in surface graft polymerization onto ePTFE through glycidyl methacrylate (GMA) as a bridge linkage following argon (Ar) plasma treatment. The epoxy group of GMA was expected to react with the peroxide groups produced on ePTFE by Ar plasma exposure, and its methacrylic groups can copolymerize with various monomers. In the present study, we selected 2-methacryloyloxyethyl phosphorylcholine (MPC) as a model monomer and the blood compatibility of modified ePTFE was evaluated. Two sequences of surface grafting were compared. In a two-step graft polymerization, GMA was first immobilized onto Ar plasma treated ePTFE, and then MPC was polymerized. In a one-step graft copolymerization, MPC and GMA were mixed and copolymerized simultaneously onto Ar plasma treated ePTFE, resulting in a poly(MPC-co-GMA) (PMG) graft surface. The roughness of the node-and-fibril structure of ePTFE was reduced by the uniform polymer layer, and the modified ePTFE had a good hydrophilic nature even after being stored in an aqueous environment for 30 days. The indispensable GMA in graft polymerization improved the surface grafting on ePTFE. The one-step and two-step graft polymerization methods could decrease the number of adhered platelets, and almost inhibit platelet activation. We concluded that graft polymerization with the GMA linker provides a novel strategy to modify the chemically inert ePTFE surfaces for functionalizing as new medical devices.

Quick nuclear transportation of siRNA and in vivo hepatic ApoB gene silencing with galactose-bearing polymeric carrier.

Since previous studies have linked the genetic mutations of Apolipoprotein B (ApoB) to the low density lipoprotein (LDL) cholesterol levels, it can be believed that the knockdown of ApoB by siRNA silencing is a useful method to reduce the cardiovascular disease. However, the spontaneous uptake of siRNA is hindered, and thus vectors are necessary to aid its transfer into the cells. Among the synthetic non-viral vectors, cationic polymers are extensively investigated as possible candidates for efficient and specific gene delivery, because they can be easily modified to get different set of properties. Therefore, in this work a set of random copolymers with different molecular weight and composition were synthesized. These vectors present 2-(dimethylamino)ethyl methacrylate, as cationic monomer, and galactose units as liver-targeting moieties. From in vitro experiments, copolymers with monomer ratio and molecular weight about 0.1 and 80kDa, respectively, showed adequate transfection capabilities and displaying good cell viability, independently of the nature of the saccharides units. However, in the in vivo experiments in C57BL/6 high-fat-fed mice, a better blood compatibility and protection against degradation leading to better transfection by the random copolymers bearing galactose units was confirmed.