Development of nanosomes using high-pressure homogenization for gene therapy.
OBJECTIVE
The aim of this project was to develop a novel lipid-based formulation suitable for gene therapy.


METHODS
Novel nanosize liposome (nanosome) formulations containing pDNA (plasmid DNA) were developed using high-pressure homogenization (HPH). The effect of lipid concentration was studied at two levels: 3 mm and 20 mm. The preformed nanosomes were incubated for 18-20 h with pDNA or pDNA/protamine sulfate (PS) complex. The physical properties of the pDNA nanosomes were compared by particle size distribution and zeta-potential measurements. Their biological properties were also compared by pDNA efficiency of encapsulation/complexation, integrity, nuclease digestion, transfection efficiency and cell cytotoxicity.


RESULTS
pDNA nanosomes prepared with 20 mM lipid (nanosomes:pDNA:PS at a ratio of 8.6:1:2) had particle sizes of 170-422 nm (90% confidence). The zeta-potential of the formulation was 49.2 +/- 1.5 mV, and the pDNA encapsulation/complexation efficiency was approximately 98%. pDNA nanosomes prepared with 3 mM lipid (nanosomes:pDNA PS at a ratio of 2.09:1:2) had particle sizes of 140-263 nm (90% confidence). The zeta-potential of this formulation was 36.4 +/- 1.2 mV, and the pDNA encapsulation/complexation efficiency was approximately 100%. However, a comparison of the efficiency of transfection indicated that pDNA nanosomes prepared with low-concentration lipids (3 mM) showed significantly higher transfection efficiency compared with the pDNA nanosomes prepared with high-concentration lipids (20 mM), as well as those prepared with Fugene-6 (a commercially available transfection reagent). This particular formulation (pDNA nanosomes, 3 mM lipids) also showed significantly less cytotoxicity compared with the other pDNA nanosome formulations.


CONCLUSIONS
To conclude, these results indicate that condensing pDNA with PS followed by subsequent complexation with low-concentration nanosomes generated from HPH can produce a pDNA nanosome formulation that will boost transfection efficiency, while minimizing cytotoxicity. This new technology appears to be an efficient tool for future commercial or large-scale manufacture of DNA delivery systems for gene therapy.
Kundu
Anup K
AK
Center for Nanomedicine and Drug Delivery, Xavier University College of Pharmacy, New Orleans, Louisiana 70125-1098, USA.
Hazari
Sidhartha
S
Chinta
Dakshinamurthy Devanga
DD
Pramar
Yashoda V
YV
Dash
Srikanta
S
Mandal
Tarun K
TK
D000818:Animals; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D051379:Mice; D053769:Nanocapsules; D010957:Plasmids; D011312:Pressure; D011479:Protamines / Q000737:chemistry; D014162:Transfection
D008055:Lipids; D008081:Liposomes; D053769:Nanocapsules; D011479:Protamines; D004247:DNA