Loading of plasmid DNA into PLGA microparticles using TROMS (Total Recirculation One-Machine System): evaluation of its integrity and controlled release properties.

Loading plasmid DNA into poly(ester) microparticles usually involves the formation of a multiple emulsion, using homogenisation techniques such as sonication or Ultra-Turrax. These procedures may negatively affect the integrity of the macromolecule and consequently its activity. The aim of this study was to prepare and evaluate DNA-loaded microparticles by TROMS (Total Recirculation One-Machine System), a new procedure that is based on the formation of a multiple emulsion by the injection of the phases under a turbulent regime. Microparticles were prepared with either Resomer) RG 502 (MP 502) or RG 756 (MP 756) and DNA loading was quantified fluorimetrically. DNA loading in MP 756 was almost twice as high as in MP 502 (510 vs. 285 ng/mg, respectively). Under both formulations, the loaded plasmid was released while maintaining its integrity for at least 24 days (MP 502) and 40 days (MP 756). Finally, the transfection efficiency was studied after injection of the microparticles (MP 502) into rat skeletal muscle and compared with naked DNA injection. Injection of naked DNA (150 microg DNA per muscle) achieved higher but variable expression levels that decreased after 3 weeks. In contrast, the muscles injected with microparticles (6.8 microg DNA per muscle) showed lower but homogeneous expression values, which were maintained for at least 3 weeks.

[1]  M. Hedley,et al.  Microspheres containing plasmid-encoded antigens elicit cytotoxic T-cell responses , 1998, Nature Medicine.

[2]  L. Lunsford,et al.  Two methods for quantifying DNA extracted from poly(lactide-co-glycolide) microspheres. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[3]  Y. Capan,et al.  Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA , 1999, Pharmaceutical Research.

[4]  J. Clegg,et al.  Poly(DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. , 1997, Vaccine.

[5]  L. Lunsford,et al.  Protective immune responses elicited in mice by immunization with formulations of poly(lactide-co-glycolide) microparticles. , 2002, Vaccine.

[6]  D. Putnam,et al.  PLGA microspheres containing plasmid DNA: preservation of supercoiled DNA via cryopreparation and carbohydrate stabilization. , 1999, Journal of pharmaceutical sciences.

[7]  G. Kwon,et al.  Encapsulation of plasmid DNA in biodegradable poly(D, L-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. , 1999, Journal of controlled release : official journal of the Controlled Release Society.

[8]  M A Tracy,et al.  Factors affecting the degradation rate of poly(lactide-co-glycolide) microspheres in vivo and in vitro. , 1999, Biomaterials.

[9]  H. Merkle,et al.  Microencapsulation of DNA using poly(DL-lactide-co-glycolide): stability issues and release characteristics. , 1999, Journal of controlled release : official journal of the Controlled Release Society.

[10]  H. Weintraub,et al.  Expression of transfected DNA depends on DNA topology , 1986, Cell.

[11]  J. Hagstrom,et al.  Plasmid DNA entry into postmitotic nuclei of primary rat myotubes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Hedley,et al.  Biological potency of microsphere encapsulated plasmid DNA. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[13]  R. Bodmeier,et al.  Somatostatin containing biodegradable microspheres prepared by a modified solvent evaporation method based on W/O/W-multiple emulsions , 1995 .

[14]  G. Farrar,et al.  Formulation of poly(D,L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[15]  Y. Hsu,et al.  Comparison of process parameters for microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic) acid microspheres. , 1999, Journal of drug targeting.