Gene Delivery Particles Research Articles

Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells.

AbstractMost in vivo gene therapies will require cell-specific targeting. Although vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing. In this study, we examine the impact of targeting two distinct endocytic routes-the caveolar and clathrin pathways-on polyethylenimine-mediated gene delivery in HeLa cells. Targeting complexes to the caveolar pathway with folic acid and the clathrin pathway with transferrin yields enhanced gene delivery relative to unmodified polyethylenimine. Colocalization studies with caveolin-1 and clathrin heavy chain indicate that the ligands successfully deliver their cargo to the intended pathways. However, inhibition of only the caveolar pathway-whether through the use of small molecule drugs or RNA interference-reduces gene delivery efficiency, suggesting that successful polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. Transfections in the presence of chloroquine and pH tracking studies suggest that a contributing factor to the success of the caveolar pathway is avoidance of lysosomes. Collectively, these data demonstrate that uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials.

The Receptor-Mediated Endocytosis of Nonspherical Particles

Enveloped viruses and nanosized biomimetic particles for drug and gene delivery enter target cells mainly through receptor-mediated endocytosis. A few models have been presented to elucidate the mechanics of particle engulfment by the cell membrane, showing how size and surface chemico-physical properties favor or oppose internalization. In this work, the effect of particle nonsphericity is addressed considering elliptical cylindrical particles with aspect ratio Γ. Using a continuum energetic approach, three different conditions have been identified: for sufficiently small Γ, the particle is not even wrapped by the cell membrane; for sufficiently large Γ, the particle is partially wrapped (“frustrated endocytosis”); and for intermediate values of Γ, the particle is fully wrapped and eventually internalized. Given the pleomorphism of viruses and the broad spectrum of shapes for nanosized biomimetic particles, the results presented may be of interest to virologists, pharmacologists, toxicologists, and nanotechnologists.

Polyethylenimine–DNA solid particles for gene delivery

Polyethylenimine (PEI), a cationic polymer, was used to develop a non-viral vector for gene delivery. A simple, reproducible process is described with which to condense plasmid DNA with PEI. When prepared at the optimum charge ratio of 6.3 ( ± ; PEI:DNA, 5:1 w/w), PEI–DNA complexes were 30–60 nm in diameter and excluded intercalating dyes from the plasmid DNA. The particles were stable for more than one month at 4°C with respect to size and transfection activity. PEI–condensed DNA transfected a broad range of murine and human tumor cell lines (B16, Lewis Lung, SK-OV-3 and LS180) in vitro in the presence of fetal calf serum. Intraperitoneal administration of PEI–condensed DNA resulted in significant gene expression in a human ovarian cancer peritoneal xenograft model.

Preparation and Characterization of Thermosensitive Submicron Particles for Gene Delivery

In this study, a new thermosensitive material was proposed as a carrier for gene delivery. The thermosensitive submicron particles were synthesized by the dispersion copolymerization of N-isopropylacylamide (NIPA) with a relatively new, cationic comonomer, N-3-dimethylaminopropylmethacrylamide (DMAPM) with higher ionization ability with respect to the commonly used cationic comonomers. To achieve particle sizes smaller than 1 microm, suitable for gene delivery, the total monomer concentration in the dispersion copolymerization was kept at a sufficiently low level. The size of poly(NIPA-co-DMAPM) particles was determined as 454 nm, by AFM in dry state. The poly(NIPA-co-DMAPM) particles showed both temperature and pH sensitivity in the aqueous media. The plasmid DNA adsorption onto the thermosensitive cationic particles was investigated at different temperatures and pHs. The adsorbed amount of plasmid DNA onto the particles was significantly increased by the introduction of cationic comonomer. The equilibrium plasmid DNA adsorptions up to 13 mg/g dry particles were achieved at physiological pH. Approximately 36% w/w of adsorbed plasmid could be desorbed from the cationic nanolatex. The results of biocompatibility studies performed with mouse fibroblast cells showed the suitability of thermosensitive cationic particles for intended application.

A Nanoparticle-Based Model Delivery System To Guide the Rational Design of Gene Delivery to the Liver. 1. Synthesis and Characterization

Nonviral gene delivery systems are amenable to forming colloidal particles with a wide range of physicochemical properties that include size, surface charge, and density and type of ligand presented. However, it is not known how to best design these particles without having a set of physicochemical design constraints that have been optimized for the intended gene delivery application. Here, a nanoparticle-based model delivery system is developed that can mimic the surface properties of nonviral gene delivery particles, and this model system is used to define design constraints that should be applied to next generation gene delivery particles. As a test case, a well-defined nanoparticle-based system is developed to guide the rational design of gene delivery to hepatocytes in the liver. The synthetic scheme utilizes monodisperse polystyrene particles and provides for variation of mean particle size and particle size distribution through variation in reaction conditions. The nanoparticles are PEGylated to provide stability in serum and also incorporate targeting ligands, e.g., galactose, at tunable densities. Four nanoparticles are synthesized from uniformly sized polystyrene beads specifically for the purpose of identifying design constraints to guide next generation gene delivery to the liver. These four nanoparticles are Gal-50 and Gal-140, that are galactosylated 50 and 140 nm nanoparticles, and MeO-50 and MeO-140, that are methoxy-terminated 50 and 140 nm nanoparticles. All four particles have the same surface charge, and Gal-50 and Gal-140 have the same surface galactose density. The availability of galactose ligands to receptor binding is demonstrated here by agglutination with RCA120.

1109. Single-Cell Observations of Intracellular Gene Delivery Particles: pH Buffering and Trafficking

For a number of polyplex formulations, enhanced gene delivery efficiency has been correlated with an inherent pH-buffering capacity or incorporation of pH-buffering components. In vitro monitoring of the intracellular pH-buffering action of these formulations has generally been studied using techniques that provide average results from populations of cells. To improve the understanding of the pH-buffering function and how it affects gene delivery, we have incorporated the pH-sensitive marker SNARF-4F in a variety of gene delivery particles and examined the in vitro intracellular trafficking of these formulations by laser scanning confocal microscopy. We are able to follow the individual vesicles in time and space within single live cells by this method and to average over many vesicles and cells to compare to previously reported results. This is the first example of spatiotemporal intracellular pH measurements of the localized environments experienced by gene delivery vehicles. We have examined polyplexes with (e.g. branched polyethylenimine) and without (e.g poly-L-lysine) pH-buffering capacity and have compared unstabilized polyplexes with those salt-stabilized through the incorporation of poly(ethylene glycol). The method applied here allows us to evaluate and describe kinetics of the various polyplexes' intracellular pH-buffering capacity and trafficking. In conjunction with quantitative assessments of nucleic acid uptake to cells and reporter gene expression, these studies offer new information on the contributions and limitations of pH-buffering components to polyplex gene delivery.

Neutral Postgrafted Colloidal Particles for Gene Delivery

Surface modification of cationic lipoplexes has been carried out by means of a postgrafting reaction. The original lipoplexes described comprise a cationic lipid, a neutral lipid, poly(ethylene glycol)-cholesterol (with or without a targeting ligand) and DNA. Modifying their surface via a chemical, postgrafting reaction did not alter their size (approximately 100 nm) nor their ability to compact DNA, but did give a reduced zeta potential (approximately 0 mV) to afford surface neutral particles. With the modified lipoplexes nonspecific NIH3T3 cell surface binding in vitro was inhibited. Intravenous injection of the neutralized lipoplexes in mice showed decreased accumulation of the particles in the lung as compared to PEGylated cationic lipoplexes. Tumor targeting was also achieved in vivo by the addition of an RGD-PEG-Cholesterol as a lipid-ligand in the postgrafted lipoplex formulation.

The fractal structure of polycation–DNA complexes

We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.

363. Uptake and Intracellular Trafficking of Stabilized and Unstabilized Gene Delivery Particles

The intracellular trafficking of gene delivery particles has primarily been studied using delivery vehicles that are not formulated for in vivo conditions. Stabilized particles appropriate for in vivo use may be processed differently than the unstabilized analogues. To address this issue, we have used transmission electron microscopy to examine the uptake and intracellular trafficking of stabilized and unstabilized polyplexes. We have used polyplexes prepared with plasmid DNA (pDNA) and either branched polyethylenimine or a linear, β-cyclodextrin-containing polycation. For both polycations, unstabilized polyplexes aggregated in solution and entered cells as large aggregates. Polyplexes stabilized by addition of poly(ethylene glycol), however, produced salt-stablized particles that were observed as discrete entities within cells. Immunolabeling with colloidal gold markers demonstrated the intracellular distribution of polyplexes and the presence of intracellular, unpackaged polyplex components in both the cytoplasm and the nucleus of cells.

PEGylation significantly affects cellular uptake and intracellular trafficking of non-viral gene delivery particles

In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear beta-cyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.

Transferrin-containing, cyclodextrin polymer-based particles for tumor-targeted gene delivery.

Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.

Modified silica particles for gene delivery

In somatic gene therapy, new concepts for the transfer of DNA into specific cell nuclei are of interest. Inorganic nanoparticles have an interesting potential as DNA carrier system due to the possibility to tailor their surface reactivity and the electrical surface potential (zeta potential) that can be obtained by the surface modification. SiO2 nanoparticles have been chosen for reasons of low toxicity. In order to obtain positively charged nanoparticles, basic surface groupings have been selected. Different types of alkoxysilanes and amines have been tested for surface modification of SiO2 nanoparticles. The zeta potential at pH=7.4 could be varied from −38.8 mV (unmodified 10 nm particles) to +20 mV (gamma-aminopropyl-triethoxysilane) and to +49.8 mV (glycidoxypropyltrimethoxysilane and ethylendiamine). DNA could be completely immobilized at the nanoparticle surface and nanoparticle/DNA ratios between 2 and 15 (w/w) were obtained. The interaction of DNA with the particle surface correlates with an increasing number of modifier molecules on the particle surface.