Stabilized Plasmid Lipid Particles Research Articles

Enzyme-responsive destabilization of stabilized plasmid-lipid nanoparticles as an efficient gene delivery

Stabilized plasmid-lipid particles (SPLPs) have been developed to overcome the low stability issue of cationic liposomes, however, SPLPs that are too stable result in unsatisfactory transfection efficiency. In this article, we prepared enzyme-responsive SPLPs (eSPLPs) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and mPEG-GLFG-K-(C16)2, a PEG lipid with an enzymatically-cleavable linker (glycine-phenylalanine-leucine-glycine, GFLG). eSPLPs were successfully prepared with plasmid DNA (pDNA) encapsulation efficiency of over 80%, using the detergent dialysis method. The PEG shell stabilized eSPLPs and maintained a hydrodynamic diameter of around 200nm. Although typical SPLPs were relatively intact in endosomal condition, the PEG shell of eSPLPs was cleaved following the degradation of the GFLG linker by cathepsin B in the endosome. Then, eSPLPs collapsed and induced endosomal disruption triggering the controlled release of the encapsulated pDNA into cytoplasm. Owing to the enzyme-responsive destabilization, eSPLPs showed a 10 to 100-fold higher transfection efficiency than control SPLPs, which was confirmed using luciferase assay. These results suggest that eSPLPs might be promising candidates for practical use as gene delivery systems, with both stability and high transfection efficiency for future in vivo applications.

The combination of stabilized plasmid lipid particles and lipid nanoparticle encapsulated CpG containing oligodeoxynucleotides as a systemic genetic vaccine

DNA vaccines offer unique potential for generating protective and therapeutic immunity against infectious and malignant diseases. Unfortunately, rapid degradation and poor cellular uptake has significantly limited the efficacy of 'naked' plasmid DNA vaccines. We have previously described stabilized plasmid lipid particles (SPLP) as effective nonviral gene delivery vehicles for the transfection of tumours at distal sites following intravenous administration. Based on their low toxicity and favourable transfection profile following systemic administration, we investigate SPLP as gene delivery vehicles for the generation of a systemically administered genetic vaccine. The uptake of SPLP and their ability to transfect splenic antigen presenting cells (APC) following systemic administration is assessed through fluorescently-labelled SPLP in combination with phenotype markers and a very sensitive flow cytometry-based assay for the detection of the transgene, beta-galactosidase. The priming of antigen-specific adaptive and humoural immune responses following vaccination with SPLP alone or in combination with liposomal nanoparticle encapsulated CpG-ODN containing oligodeoxynucleotides (LN CpG-ODN) is characterized through the use of antigen-specific cytotoxicity assays, interferon-gamma secretion assays and enzyme-linked immunosorbant assay. We demonstrate that SPLP are taken up by and transfect APC in the spleen following intravenous administration and that, in the presence of a strong immunostimulatory signal provided by LN CpG-ODN, are able to prime transgene-specific humoural and cellular immune responses. SPLP represent an effective candidate for the nonviral delivery of a systemic genetic vaccine when combined with additional immune stimulation provided by LN CpG-ODN.

Targeting of stabilized plasmid lipid particles to hepatocytes in vivo by means of coupled lactoferrin

For non-viral gene delivery we prepared stabilized plasmid lipid particles (SPLPs), to which lactoferrin (LF) was coupled as a hepatocyte specific targeting ligand. LF–SPLPs and untargeted SPLPs labeled with [3H]cholesteryloleyl-ether were injected into rats. About 87% of the LF–SPLPs were eliminated from the blood within 5 min, while 80% of untargeted SPLPs were still circulating after 2 h. Fifty-two percent of the LF–SPLPs were taken up by hepatocytes, while non-parenchymal liver cells accounted for 16% of the uptake. Despite the efficient targeting of LF–SPLPs to hepatocytes and their capacity to transfect HepG2 and COS-7 cells in vitro, expression of a reporter gene was not detected in vivo. Overall, covalent coupling of LF to SPLPs leads to massive delivery in hepatocytes after systemic administration. However, these LF–SPLPs are not able to transfect these cells in vivo.

Calcium enhances the transfection potency of stabilized plasmid–lipid particles

Previous work from this laboratory has shown that plasmid DNA can be encapsulated in small (70-nm-diameter) stabilized plasmid–lipid particles (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle. SPLP preferentially transfect tumor tissue following intravenous administration. Although the levels of transgene expression in vivo are greater for SPLP than can be achieved with naked DNA or complexes, they are lower than may be required for therapeutic benefit. In the present work we examine whether Ca 2+ can enhance the transfection potency of SPLP. It is shown that Ca 2+ can enhance SPLP transfection potency in bovine hamster kidney cells by 60- to 100-fold when treated in serum containing medium and an additional 60-fold when serum is absent for the initial 10 min of the transfection period. When cells are treated with SPLP in the presence of Ca 2+, there is a fivefold increase in intact plasmid in the cell. It is also shown that this Ca 2+ effect involves the formation of calcium phosphate precipitates; however, these precipitates are not directly associated with the SPLP plasmid DNA. The ability of calcium phosphate to facilitate delivery of other macromolecules without direct association is also demonstrated by the release of large-molecular-weight dextrans from endosomal/lysosomal compartments in the presence of calcium phosphate. Finally, it is shown that, unlike naked DNA, SPLP transfection potency in the presence of calcium phosphate is not affected by nuclease activity.

A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA

A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy. 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. 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. 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.

Stabilized plasmid–lipid particles containing PEG-diacylglycerols exhibit extended circulation lifetimes and tumor selective gene expression

Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small (∼100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG–lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG–diacylglycerol lipids (PEG–S-DAGs). SPLP were prepared incorporating PEG–dimyristoylglycerol (C 14), PEG–dipalmitoylglycerol (C 16) or PEG–distearoylglycerol (C 18) and the rate of PEG–lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG–lipid component and circulation lifetime. PEG–S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG–distearoylglycerol (C 18) containing SPLP bypass so-called ‘first pass’ organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG–S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.

Distal Cationic Poly(Ethylene Glycol) Lipid Conjugates in Large Unilamellar Vesicles Prepared by Extrusion Enhance Liposomal Cellular Uptake

Cationic poly(ethylene glycol)‐lipid conjugates (CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine (DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively (CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG‐containing LUVs and SPLP (stabilized plasmid‐lipid particles), have shown significant increases in the binding of CPL‐LUV to cells, and have observed dramatically enhanced transfection (up to a million‐fold) of cells with CPL‐SPLP in the presence of calcium [Chen et al. (2000) Bioconjugate Chem. 11, 433–437; Fenske et al. (2001) Biochim. Biophys. Acta 1512, 259–272; Palmer et al. (2003) Biochim. Biophys. Acta 1611, 204–216]. In the present study, we examine a variety of CPL properties (such as polarity and CMC) and characterize CPL‐vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1 (1.3 µM) to CPL8 (2 µM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles (LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes (DSPC/Chol, 55:45) at 60°C. The binding of CPL‐LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells (as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic (plasmid and antisense) drugs.

Transfection properties of stabilized plasmid–lipid particles containing cationic PEG lipids

Recent work has shown that plasmid DNA can be efficiently encapsulated in well-defined “stabilized plasmid–lipid particles” (SPLP) that have potential as systemic gene therapy vehicles [Gene Ther. 6 (1999) 271]. In this work, we examine the influence of ligands that enhance cellular uptake on the transfection potency of SPLP. The ligand employed is a cationic poly(ethylene glycol) (PEG) lipid (CPL) consisting of a lipid anchor and a PEG 3400 spacer chain with four positive charges at the end of the PEG (CPL 4). It is shown that up to 4 mol% CPL 4 can be inserted into preformed SPLP, resulting in up to 50-fold enhancements in uptake into baby hamster kidney (BHK) cells. The addition of Ca 2+ to SPLP-CPL 4 (CPL 4-incorporated SPLP) results in up to 10 6-fold enhancements in transgene expression, as compared to SPLP in the absence of either CPL 4 or Ca 2+. These transfection levels are comparable to those observed for plasmid DNA–cationic lipid complexes (lipoplexes) but without the cytotoxic effects noted for lipoplex systems. It is concluded that in the presence of Ca 2+ and appropriate ligands to stimulate uptake, SPLP are highly potent transfection agents.

Stabilized Plasmid–Lipid Particles: Pharmacokinetics and Plasmid Delivery to Distal Tumors following Intravenous Injection

A previous study has shown that plasmid DNA can be encapsulated in lipid particles (SPLP, “stabilized plasmid lipid particles”) of approximately 70 nm diameter composed of 1,2-dioleoyl-3-phosphatidyl-ethanolamine (DOPE), the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) conjugated to ceramide (PEG-Cer) using a detergent dialysis process (Wheeler et al. (1999) Gene Therapy 6, 271-281). In this work we evaluated the potential of these SPLPs as systemic gene therapy vectors, determining their pharmacokinetics and the biodistribution of the plasmid and lipid components. It is shown that the blood clearance and the biodistribution of the SPLPs can be modulated by varying the acyl chain length of the ceramide group used as lipid anchor for the PEG polymer. Circulation lifetimes observed for SPLPs with PEG-CerC14 and PEG-CerC20 were t1/2 = ± 1 and ± 10 h, respectively. The SPLPs are stable while circulating in the blood and the encapsulated DNA is fully protected from degradation by serum nucleases. The accelerated clearance of SPLPs with PEG-CerC14 is accompanied by increased accumulation in liver and spleen as compared to PEG-CerC20 SPLPs. Delivery of intact plasmid to liver and spleen was detected. Significant accumulation (approximately 10% of injected dose) of the long circulating SPLPs with PEG-CerC20 in a distal tumor (Lewis lung tumor in the mouse flank) was observed following iv application and delivery of intact plasmid to tumor tissue at approximately 6% injected dose/g tissue is demonstrated.

Cationic lipid-mediated transfection of cells in culture requires mitotic activity.

Cationic lipid-based delivery systems such as lipoplexes or stabilized plasmid-lipid particles (SPLP) represent a safer alternative to viral systems for gene therapy applications. We studied the impact of cell cycle status on the efficiency of transfection of human ovarian carcinoma tumor cells using two cationic-lipid based delivery systems. Cells arrested in the G1 phase of the cell cycle by treatment with aphidicolin were compared with an asynchronous dividing population of cells. Treatment of the cells with aphidicolin had no effect on the rate of internalization of the lipid formulated DNA or on the level of gene expression observable in stably transfected cells. However, cells treated with aphidicolin exhibited 20-fold lower reporter gene activity than asynchronous control cells upon incubation with lipoplexes. When cells arrested in the G1 phase were allowed to proceed through the cell cycle in the presence of the lipoplex or SPLP, transgene expression was found to coincide with the transition of cells from the G2/M phase into the G1 phase of the subsequent cell cycle. In addition, higher levels of reporter gene expression were observed when the cells were incubated with lipoplexes or SPLP during, or just before, mitosis. These results suggest that it may be possible to augment cationic lipid-mediated transfection by manipulating the cell cycle status of the target cells.