ABSTRACT
Using a new controlled mixing process, highly transfection-competent polyplexes were formed and subsequently encapsulated within a lipid bilayer. The resulting "pre-condensed stable plasmid lipid particles" (pSPLPs) have small size (104+/-3 nm) and low surface charge characteristics. The formulation process equally enabled lipid encapsulation of either poly-L-lysine or poly(ethyleneimine) (PEI) condensed DNA, and the endosomolytic benefits of PEI were demonstrated in in vitro gene expression studies. The clearance properties of pSPLP were compared to similar formulations with an uncondensed payload (SPLP) in A/J mice bearing subcutaneous Neuro-2a tumors. Plasma clearance of pSPLP (t(1/2)=6.6 h) was similar to SPLP (t(1/2)=7.1 h), allowing significant accumulation at distal tumor target sites. Gene expression profiles were evaluated in vivo using the Neuro-2a model, and PEI-pSPLP formulations demonstrated a sixfold increase in reporter gene expression in tumors compared to SPLP. No significant gene expression was observed in the liver, lung, or spleen when mice were treated with either SPLP or pSPLP, and both formulations were equally well tolerated. The results support the lipid encapsulation of polyplex plasmid DNA as a means of changing its pharmacologic properties and enabling systemic delivery. The inclusion of endosomolytic DNA-condensing agents such as PEI greatly improves the potency of SPLP.
Subject(s)
Genetic Vectors , Plasmids/administration & dosage , Plasmids/genetics , Animals , Cell Line, Tumor , Cryoelectron Microscopy , Gene Expression , Genetic Therapy/methods , In Vitro Techniques , Liposomes , Male , Mice , Mice, Inbred A , Neuroblastoma/genetics , Neuroblastoma/therapy , TransfectionABSTRACT
PURPOSE: A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy. METHODS: Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution. RESULTS: Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongated morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O. CONCLUSIONS: This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.
