Evaluation of Pharmacokinetics of Bioreducible Gene Delivery Vectors by Real-time PCR
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To investigate pharmacokinetics of reversibly stabilized DNA nanoparticles (rSDN) using a single-step lysis RT-PCR.
rSDN were prepared by coating bioreducible polycation/DNA polyplexes with multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. Targeted polyplexes were formulated by linking cyclic RGD ligand (c(RGDyK)) to the HPMA surface layer of rSDN. The pharmacokinetic parameters in tumor-bearing mice were analyzed by PKAnalyst®.
The pharmacokinetics of naked plasmid DNA, simple DNA polyplexes, rSDN, and RGD-targeted rSDN exhibited two-compartment model characteristics with area under the blood concentration–time curve (AUC) increasing from 1,102 ng∙ml−1∙min−1 for DNA to 3,501 ng∙ml−1∙min−1 for rSDN. Non-compartment model analysis revealed increase in mean retention time (MRT) from 4.5 min for naked DNA to 22.9 min for rSDN.
RT-PCR is a sensitive and convenient method suitable for analyzing pharmacokinetics and biodistribution of DNA polyplexes. Surface stabilization of DNA polyplexes can significantly extend their MRT and AUC compared to naked DNA. DNA degradation in rSDN in blood circulation, due to a combined effect of disulfide reduction and competitive reactions with charged molecules in the blood, contributes to DNA elimination.
KEY WORDSgene delivery pharmacokinetics real-time PCR tumor targeting
Area under the blood concentration–time curve
Liquid chromatography–mass spectroscopy
Mean retention time
Reversibly-stabilized DNA nanoparticles, i.e. rPLL/DNA complexes coated with HPMA copolymer
Real-time polymerase chain reaction
This research was financially supported by the National Institutes of Health (CA 109711). We thank Drs. Amjad and Cha of the Anti-Infective Research Laboratory of Wayne State University for help with RT-PCR.
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