A Fibrin Glue Composition as Carrier for Nucleic Acid Vectors

作者：Schillinger Ulrike; Wexel Gabriele; Hacker Christian; Kullmer Martin; Koch Christian; Gerg Michael; Vogt Stephan; Ueblacker Peter; Tischer Thomas; Hensler Daniel; Wilisch Jonas; Aigner Joachim; Walch Axel; Stemberger Axel; Plank Christian^*

来源：Pharmaceutical Research, 2008, 25(12): 2946-2962.

DOI：10.1007/s11095-008-9719-8

• gene-activated matrix fibrinogen
• gene therapy
• gene transfer
• tissue adhesive
• tissue engineering

Purpose. Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration.
Methods. Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL (R) and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally, chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model.
Results. The optimized glue is based on the fibrinogen component of TISSUCOL (R), a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (> 70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group).
Conclusions. COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.

• 出版日期2008-12