Scherer Franz, Schillinger Ulrike, Putz Ursula, Stemberger Axel, Plank Christian
Institute of Experimental Oncology, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany.
J Gene Med. 2002 Nov-Dec;4(6):634-43. doi: 10.1002/jgm.298.
Naked DNA and standard vectors have previously been used for gene delivery from implantable carrier matrices with great potential for gene therapeutic assistance of wound healing or tissue engineering. We have previously developed copolymer-protected gene vectors which are inert towards opsonization. Here we examine their potency in carrier-mediated gene delivery in comparison to standard vectors using a vector-loaded collagen sponge model.
Equine collagen type I sponges were loaded by a lyophilization method with naked DNA, polyethylenimine (PEI)-DNA, DOTAP/cholesterol-DNA and copolymer-protected PEI-DNA. These preparations were characterized in terms of vector-release, cell growth on the matrices and reporter gene expression by cells colonizing the sponges in vitro and in vivo. Subcutaneous implantation of sponges in rats served as an in vivo model.
At the chosen low vector dose, the loading efficiency was at least 86%. Naked DNA-loaded collagen matrices lost 77% of the DNA dose in an initial burst in aqueous buffer in vitro. The other preparations examined displayed a sustained vector release. There was no difference in cell growth and invasion of the sponges between vector-loaded and untreated collagen grafts. Reporter gene expression from cells colonizing the sponges in vitro was observed for not more than 7 days with naked DNA, whereas the lipoplex and polyplex preparations yielded long-term expression throughout the experimental period of up to 56 days. The highest expression levels were achieved with the PEI-DNA-PROCOP (protective copolymer) formulation. Upon subcutaneous implantation in rats, no luciferase expression was detected with naked DNA preparations. DOTAP/cholesterol-DNA and PEI-DNA-loaded implants lead to reporter gene expression for at least 3 days, but with poor reproducibility. PEI-DNA-PROCOP collagen matrices yielded consistently the highest reporter gene expression levels for at least 7 days with good reproducibility.
With the preparation method chosen, lipoplex- and polyplex-loaded collagen sponges are superior in mediating sustained gene delivery in vitro and local transfection in vivo as compared to naked DNA-loaded sponges. Protective copolymers are particularly advantageous in promoting the tranfection capacity of polyplex-loaded sponges upon subcutaneous implantation, likely due to their stabilizing and opsonization-inhibiting properties.
裸DNA和标准载体先前已被用于从可植入载体基质进行基因递送，在伤口愈合或组织工程的基因治疗辅助方面具有巨大潜力。我们先前开发了对调理作用呈惰性的共聚物保护基因载体。在此，我们使用载有载体的胶原海绵模型，将它们与标准载体相比，研究其在载体介导的基因递送中的效力。
通过冻干法将裸DNA、聚乙烯亚胺（PEI）-DNA、DOTAP/胆固醇-DNA和共聚物保护的PEI-DNA负载到I型马胶原海绵中。通过载体释放、基质上的细胞生长以及在体外和体内定殖于海绵的细胞的报告基因表达，对这些制剂进行表征。将海绵皮下植入大鼠作为体内模型。
在选定的低载体剂量下，负载效率至少为86%。载有裸DNA的胶原基质在体外水性缓冲液中的初始突释中损失了77%的DNA剂量。所检测的其他制剂显示出持续的载体释放。负载载体的胶原移植物与未处理的胶原移植物在海绵的细胞生长和侵袭方面没有差异。对于裸DNA，在体外定殖于海绵的细胞中观察到报告基因表达不超过7天，而脂质体和多聚体制剂在长达56天的整个实验期间产生长期表达。PEI-DNA-PROCOP（保护性共聚物）制剂实现了最高表达水平。在大鼠皮下植入后，裸DNA制剂未检测到荧光素酶表达。载有DOTAP/胆固醇-DNA和PEI-DNA的植入物导致报告基因表达至少3天，但重现性差。PEI-DNA-PROCOP胶原基质在至少7天内始终产生最高的报告基因表达水平，且重现性良好。
采用所选的制备方法，与载有裸DNA的海绵相比，载有脂质体和多聚体的胶原海绵在体外介导持续基因递送和体内局部转染方面更具优势。保护性共聚物在促进皮下植入后载有多聚体的海绵的转染能力方面特别有利，这可能归因于它们的稳定和抑制调理作用的特性。
