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Sterilization of poly(dimethylamino) ethyl methacrylate-based gene transfer complexes
G W Bos1, A Trullas-Jimeno, W Jiskoot
1Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, P.O. Box 80.082, 3508 TB, Utrecht, The Netherlands. g.w.bos@pharm.uu.nl
International Journal of Pharmaceutics
|January 4, 2001
Summary
Sterile filtration effectively sterilizes polyplexes for gene therapy without compromising DNA integrity or transfection potential. Autoclaving, however, damages polyplexes, rendering them ineffective for gene delivery.
Area of Science:
- Biotechnology
- Gene Therapy
- Materials Science
Background:
- Parental administration of polyplex formulations for gene therapy or genetic vaccination necessitates sterile preparations.
- Poly(2-(dimethylamino) ethyl methacrylate) (pDMAEMA) based polyplexes are utilized as gene transfer agents.
Purpose of the Study:
- To assess the feasibility and limitations of autoclaving and sterile filtration for sterilizing pDMAEMA-based polyplexes.
- To evaluate the impact of sterilization methods on DNA integrity, polyplex characteristics, and transfection potential.
Main Methods:
- Agarose gel electrophoresis and circular dichroism spectroscopy were employed to analyze DNA topology and integrity.
- Transfection potential was assessed in COS-7 and OVCAR-3 cell lines.
- Sterilization methods included autoclaving, sterile filtration, and a combination of both.
Main Results:
- Sterile filtration preserved DNA topology and integrity, with retained transfection potential in cells.
- Autoclaving significantly altered polyplex characteristics, leading to a complete loss of transfection capability.
- Material loss during filtration could be minimized by pre-coating filters with polyplexes.
- Sterile filtration of 'naked' DNA and separate sterilization of polymer and DNA were also feasible.
Conclusions:
- Sterile filtration is a viable method for sterilizing pDMAEMA-based polyplexes for gene therapy applications.
- Careful consideration of filter type and volume is necessary to minimize material loss during filtration.
- Alternative strategies, such as separate sterilization of components, can be employed.