Lipid-based Nanoparticles for Nucleic Acid Delivery

Abstract Lipid-based colloidal particles have been extensively studied as systemic gene delivery carriers. The topic that we would like to emphasize is the formulation/assembly of lipid-based nanoparticles (NP) with diameter under 100 nm for delivering nucleic acid in vivo. NP are different from cationic lipid–nucleic acid complexes (lipoplexes) and are vesicles composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The diameter of the NP is an important attribute to enable NP to overcome the various in vivo barriers for systemic gene delivery such as: the blood components, reticuloendothelial system (RES) uptake, tumor access, extracellular matrix components, and intracellular barriers. The major formulation factors that impact the diameter and encapsulation efficiency of DNA-containing NP include the lipid composition, nucleic acid to lipid ratio and formulation method. The particle assembly step is a critical one to make NP suitable for in vivo gene delivery. NP are often prepared using a dialysis method either from an aqueous-detergent or aqueous-organic solvent mixture. The resulting particles have diameters about 100 nm and nucleic acid encapsulation ratios are >80%. Additional components can then be added to the particle after it is formed. This ordered assembly strategy enables one to optimize the particle physico-chemical attributes to devise a biocompatible particle with increased gene transfer efficacy in vivo. The components included in the sequentially assembled NP include: poly(ethylene glycol) (PEG)-shielding to improve the particle pharmacokinetic behavior, a targeting ligand to facilitate the particle–cell recognition and in some case a bioresponsive lipid or pH-triggered polymer to enhance nucleic acid release and intracellular trafficking. A number of groups have observed that a PEG-shielded NP is a robust and modestly effective system for systemic gene or small interfering RNA (siRNA) delivery.     

Novel Liposomal Formulation for Targeted Gene Delivery

Purpose Development of a polyethylene glycol (PEG)-stabilized immunoliposome (PSIL) formulation with high DNA content suitable for in vivo intravenous administration and targeted gene delivery. Materials and Methods Plasmid DNA was condensed using 40% ethanol and packaged into neutral PSILs targeted to the mouse transferrin receptor using monoclonal antibodies (MAbs; clones RI7 and 8D3) attached to their PEG maleimide moieties. PSILs size was measured by quasi-elastic light scattering. The targeting capacity of the formulation was determined by transfection of mouse neuroblastoma Neuro 2A (N2A) cells with PSIL-DNA complexes conjugated with either RI7 or 8D3 MAbs. Results DNA encapsulation and MAb conjugation efficiencies averaged 71 ± 14% and 69 ± 5% (mean ± SD), respectively. No alteration in mean particle size (< 100 nm) or DNA leakage were found after 48 h storage in a physiological buffer, and the in vivo terminal half-life reached 23.9 h, indicating that the PSIL-DNA formulation was stable. Addition of free RI7 MAbs prevented transfection of N2A cells with PSIL-DNA complexes conjugated with either RI7 or 8D3 MAbs, confirming that the transfection was transferrin receptor-dependent. Conclusions The present data suggest that our new PSIL formulation combines molecular features required for targeted gene therapy including high DNA encapsulation efficiencies and vector-specific transient transfection capacity. Rivest, Phivilay, contributed equally to this work.     

Cationic Polymer Based Gene Delivery Systems

Gene transfer to humans requires carriers for the plasmid DNA which canefficiently and safely carrythe gene into the nucleus of the desired cells. A series of chemically differentcationic polymers arecurrently being investigated for these purposes. Although many cationic polymersindeed condense DNAspontaneously, which is a requirement for gene transfer in most types of cells,the physicochemical andbiopharmaceutical behavior of the current generation of polyplexes severelylimits an efficient genetransfer in vitro and especially in vivo. This papersummarizes recent physicochemical and biologicalinformation on polyplexes and aims to provide new insights with respect to thistype of gene deliverysystem. Firstly, the chemical structure of frequently studied cationic polymersis represented. Secondly,the parameters influencing condensation of DNA by cationic polymers aredescribed. Thirdly, the surfaceproperties, solubility, aggregration behavior, degradation and dissociation ofpolyplexes are considered.The review ends by describing the in vitro and in vivo genetransfection behavior of polyplexes.     

乳剂用作基因给药载体的研究进展

基因治疗是目前研究的热点,带正电阳离子乳剂则是一种安全、高效的新型基因给药载体,其通过静电引力与DNA形成带正电的复合体后与细胞膜结合,利用乳剂与细胞膜的相容性将目的基因转运到细胞内而达到基因治疗的目的。综述带正电阳离子乳剂的组成,以及带正电阳离子乳剂-DNA复合物的制备和应用研究进展。     

Synthetic Glycopeptide-Based Delivery Systems for Systemic Gene Targeting to Hepatocytes

Purpose. To design, synthesize, and test synthetic glycopeptide-baseddelivery systems for gene targeting to hepatocytes by systemicadministration.Methods. All peptides were synthesized by the solid phase methoddeveloped using Fmoc chemistry on a peptide synthesizer. The bindingof galactosylated peptides to HepG2 cells and accessibility of thegalactose residues on particle surface was demonstrated by acompetition assay using ¹²⁵I-labeleld asialoorosomucoid and RCA lectinagglutination assay, respectively. DNA plasmid encoding chloramphenicolacetyl transferase (CAT) gene was complexed with a tri-galactosylatedpeptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in thepresence of an endosomolytic peptide (GM225.1) or endosomolyticlipopeptide (GM227.3) to obtain DNA particles of 100–150 nm insize. The plasmid/peptide complexes were added to HepG2 cell culturesor intravenously administered by tail vein injection into normal miceor rats. Plasmid uptake and expression was quantified by qPCR andELISA, respectively.Results. Multiple antennary glycopeptides that have the ability tocondense and deliver DNA plasmid to hepatocytes were synthesized andcomplexed with DNA plasmid to obtain colloidally stable DNA/peptidecomplexes. Addition of DNA/GM245.3/GM225.1 peptide complexes(1:3:1 (–/+/–)) to HepG2 cell cultures yielded CAT expression intransfected cells. The transfection efficiency was significantly reducedin the absence of galactose ligand or removal of endosomolytic peptide.Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5(–/+)) into the tail vein of normal rats yielded DNA uptake in theliver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3resulted in more stable DNA particles, and a 10-fold enhancement inliver plasmid uptake. CAT expression was detectable in liver followingintravenous administration of DNA/GM246.3 complexes. Addition ofendosomolytic lipopeptide GM227.3 into the complexes(DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–))) yielded a 5-fold increase inCAT expression. Liver expression was 8-fold and 40-fold higher thanlung and spleen, respectively, and localized in the hepatocytes only.The transfection efficiency in liver was enhanced by increasing DNAdose and injection volume. The plasmid uptake and expression in liverusing DNA/GM246.3/GM227.3 complexes was 100-200-fold higherthan DNA formulated in glucose. Tissue examination and serumbiochemistry did not show any adverse effect of the DNA/GM246.3/GM227.3 (1:0.5:1 (–/+/–)) complexes after intravenous delivery.Conclusions. Gene targeting to hepatocytes was achieved by systemicadministration of a well-tolerated synthetic glycopeptide-baseddelivery system. The transfection efficiency of this glycopeptide deliverysystem was dependent on peptide structure, endosomolytic activity,colloidal particle stability, and injection volume.     

Development of the Liver- and Lobe-Selective Nonviral Gene Transfer Following the Instillation of Naked Plasmid DNA Using Catheter on the Liver Surface in Mice

Purpose. The present study has undertaken the liver- and lobe-selective nonviral gene transfer following the instillation of naked plasmid DNA (pDNA) using catheter on the liver surface in mice. Methods. The polyethernylon catheter was inserted intraperitoneally through the abdominal wall and was retained on the surface of the liver right and left medial lobes. pDNA was administered through the catheter to the liver right and left medial lobes. Results. The luciferase levels produced in the applied liver lobes at 6 h after liver surface instillation of pDNA were significantly higher than those produced in other liver lobes and other tissues assayed, and ranged from approximately 5 folds higher in other lobes to 20-30 folds higher in other tissues. Following liver surface instillation of pDNA at a time from 2 to 24 h or at a volume from 15 to 60 l, the gene expressions of the applied liver lobes were always significantly higher than those of other liver lobes and other tissues. Conclusion. We have demonstrated the liver- and lobe-selective gene transfection following the instillation of naked pDNA using catheter on the liver surface in mice.     

New Cationic Lipid Formulations for Gene Transfer

Purpose. To develop appropriate dosage forms of DNA for gene delivery. Methods. 3[N-(N, N dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) was mixed either with Tween 80 alone, or with additional lipid components including castor oil and phosphatidylcholine (PC) or dioleoylphosphatidylethanolamine (DOPE) to make different lipid formulations. The particle size and the physical stability of the formulations upon mixing with plasmid DNA containing the luciferase cDNA were examined using laser light scattering measurement. The transfection activity of the DNA/lipid complexes was tested in presence or absence of serum using a cell culture system. Results. We demonstrated that many favorable properties as a gene carrier could be achieved by formulating DNA into new dosage forms using Tween 80 as the major emulsifier. Compared to the cationic liposomes, these new formulations transfected different cell lines with an equivalent or higher efficiency. Not only are they resistant to serum, but also form stable DNA complexes which could be stored for longer periods of time without losing transfection activity. Conclusions. Cationic lipids formulated into different lipid formulations using Tween 80 as a surfactant appeared to have more favorable physical and biological activities than traditional cationic liposomes as a carrier for gene delivery.     

精蛋白在改进非病毒载体基因转染中的应用

非病毒基因载体的出现,为基因治疗提供了低毒、易于大规模制备的载体。但与病毒载体相比,非病毒基因载体的转染效率仍然偏低,阻碍了非病毒基因载体的临床应用。本文旨在探讨精蛋白在改进非病毒基因载体方面的应用,希望通过合理的载体设计与优化,制备出一种高效、低毒的基因载体。     

Polyvinyl Derivatives as Novel Interactive Polymers for Controlled Gene Delivery to Muscle

Purpose. DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. Methods. A DNA plasmid, containing a CMV promoter and a -galactosidase reporter gene (CMV--gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier-Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 µl) were injected into rat tibialis muscles after surgical exposure. Immuno-histochemistry for -gal was used to visualize the sites of expression in muscle. Results. -gal expression using pDNA in saline reached a plateau while -gal expression using PVP formulations increased linearly in the dose range studied (12.5–150 µg pDNA injected) and resulted in an increase in the number and distribution of cells expressing -gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. Conclusions. Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.     

Engineered Polyallylamine Nanoparticles for Efficient In Vitro Transfection

Purpose Cationic polymers (i.e. polyallylamine, poly-L-lysine) having primary amino groups are poor transfection agents and possess high cytotoxicity index when used without any chemical modification and usually entail specific receptor mediated endocytosis or lysosomotropic agents to execute efficient gene delivery. In this report, primary amino groups of polyallylamine (PAA, 17 kDa) were substituted with imidazolyl functions, which are presumed to enhance endosomal release, and thus enhance its gene delivery efficiency and eliminate the requirement of external lysosomotropic agents. Further, systems were cross-linked with polyethylene glycol (PEG) to prepare PAA-IAA-PEG (PIP) nanoparticles and evaluated them in various model cell lines. Materials and Methods The efficacy of PIP nanoparticles in delivering a plasmid encoding enhanced green fluorescent protein (EGFP) gene was assessed in COS-1, N2a and HEK293 cell lines, while their cytotoxicity was investigated in COS-1 and HEK293 cell lines. The PAA was chemically modified using imidazolyl moieties and ionically cross-linked with PEG to engineer nanoparticles. The extent of substitution was determined by ninhydrin method. The PIP nanoparticles were further characterized by measuring the particle size (dynamic light scattering and transmission electron microscopy), surface charge (zeta potential), DNA accessibility and buffering capacity. The cytotoxicity was examined using the MTT method. Results In vitro transfection efficiency of synthesized nanoparticles is increased up to several folds compared to native polymer even in the presence of serum, while maintaining the cell viability over 100% in COS-1 cells. Nanoparticles possess positive zeta potential between 5.6–13 mV and size range of 185–230 nm in water. The accessibility experiment demonstrated that nanoparticles with higher degree of imidazolyl substitution formed relatively loose complexes with DNA. An acid-base titration showed enhanced buffering capacity of modified PAA. Conclusions The PIP nanoparticles reveal tremendous potential as novel delivery system for achieving improved transfection efficiency, while keeping the cells at ease.     

Pharmaceutical Evaluation of Gas-Filled Microparticles as Gene Delivery System

Purpose. To produce and characterize a nonviral ultrasound-controlled release system of plasmid DNA (pDNA) encapsulated in gas-filled poly(D,L-lactide-co-glycolide) microparticles (PLGA-MPs). Methods. Different cationic polymers were used to form pDNA/polymer complexes to enhance the stability of pDNA during microparticle preparation. The physico-acoustical properties of the microparticles, particle size, pDNA integrity, encapsulation efficiency and pDNA release behavior were studied in vitro. Results. The microparticles had an average particle size of around 5 m. More than 50% of all microparticles contained a gas core, and when exposed to pulsed ultrasound as used for color Doppler imaging create a signal that yields typical color patterns (stimulated acoustic emission) as a result of the ultrasound-induced destruction of the microparticles. Thirty percent of the pDNA used was successfully encapsulated and approximately 10% of the encapsulated pDNA was released by ultrasound within 10 min. Conclusions. Plasmid DNA can be encapsulated in biodegradable gas-filled PLGA-MPs without hints for a structural disintegration. A pDNA release by ultrasound-induced microparticle-destruction could be shown in vitro.     

疫苗和基因给药系统的研究进展

简述了当前疫苗和基因给药系统的研究进展，包括注射、口服、鼻腔和透皮等给药系统，并介绍了以转基因植物作为疫苗以及基因给药系统的病毒载体和非病毒载体系统。     

Novel Pentablock Copolymers for Selective Gene Delivery to Cancer Cells

Purpose  In this study, the novel poly(diethylaminoethylmethacrylate) (PDEAEM)/Pluronic F127 pentablock copolymers were found to be able to mediate high-efficiency transfection of human epithelial ovarian carcinoma (SKOV3) cell line while showing significantly lower efficacy in human epithelial retinal (ARPE-19) cell line and Swiss Mouse Fibroblast (3T3) cell line. Methods  The intracellular routes of polyplexes were investigated by confocal microscopy after appropriately labeling the polymer and DNA. Results  It was found that lesser nuclear entry in the ARPE-19 cells may result in the lower efficiency of transfection. Since the SKOV3 proliferation rate was found to be much higher than that of the ARPE-19 cells, the nuclear entry of polyplexes was assumed to be correlated with the proliferation rate, and it was hypothesized that the novel pentablock copolymers could mediate gene delivery selectively in fast growing cells. The different intracellular barriers to gene transfer may also account for the observed difference of transfection efficacy. Conclusions  Although the validity of the hypothesis that our pentablock copolymer could selectively transfect hyperproliferative cells needs further examination, this present work provides a new perspective to design targeting vectors for cancer therapies based on different characteristics among specific cell types. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effective Transfection of Rabies DNA Vaccine in Cell Culture Using an Artificial Lipoprotein Carrier System

PURPOSE: To evaluate the transfection efficiency in cell culture of rabies plasmid DNA vaccine carried by a novel artificial lipoprotein system. METHODS: Phospholipid nanoemulsions resembling the lipid core of natural lipoproteins were prepared. The artificial lipoprotein carrier system for DNA was constructed by assembling of the nanoemulsion (NE)-palmitoyl-poly-L-lysine (p-PLL)-rabies DNA complex. Agarose gel electrophoresis, zeta potential, and mobility measurement were conducted to determine the surface charge balance in various complex compositions. Transfection and transfection efficiency were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: The artificial lipoprotein system was successfully constructed and the rabies DNA vaccine was effectively transfected in glioma cell line SF-767. The amount of p-PLL incorporated into the artificial lipoprotein formulations had a significant effect on transfection efficiency. The new system also proved to be more efficient in cellular transfection of rabies DNA vaccine than the commercial lipofectamine formulation. CONCLUSIONS: Effective transfection of rabies DNA vaccine in cell culture can be achieved using the novel artificial lipoprotein carrier system, and the charge balance of the NE-p-PLL-DNA complex appears an important factor.     

Gene Transfer In Vitro and In Vivo by Cationic Lipids Is Not Significantly Affected by Levels of Supercoiling of a Reporter Plasmid

Purpose. It is a common preconception that supercoiledplasmid DNA is more desirable for the transfection of cells that the relaxedform of the plasmid. This notion has led to the recommendation that aspecification for the minimum amount of plasmid in the supercoiled formshould exist in a gene therapy product. We have tested this notion byexamining the effects of the degree of supercoiling on cationiclipid-mediated gene transfer in vitro and in vivo. Methods. An ion-exchange high performance liquidchromatography (HPLC) method was developed to accurately quantitatethe relative amounts of supercoiled DNA in purified plasmid. A sample of thepurified plasmid was fully relaxed using topoisomerase. Next, the ability ofvarious levels of supercoiled plasmid to transfect mammalian cells wasmeasured. Results. This study suggests that there is no relationbetween the degree of supercoiling and lipofection efficiency. Subsequenttransfection using several different lipofection agents, different celltypes, and an in vivo model support these results. Conclusions. In considering a specification for the amountof supercoiled plasmid in a gene therapy product, it must be noted that therelaxed forms of the plasmid are no less efficient at gene delivery than thesupercoiled forms.     

Sphingosine-Based Liposome as DNA Vector for Intramuscular Gene Delivery

Purpose. The aim of this study was to develop a labile sphingosine-based liposome for intramuscular gene delivery. Methods. Sphingosine-based liposomes were formulated in a range of solutions with phosphatidylcholine, then were associated to DNA. The physico-chemical characteristics of the sphingosine/EPC liposomes and sphingosine/EPC/DNA lipoplexes were determined. DNA stability within sphingosine-based liposomes was evaluated in the presence of a nuclease and mouse serum. In vivo gene transfer was studied by intramuscular injection with and without the electrotransfer technique. Results. By increasing the charge ratios, colloidally stable sphingosine/DNA particles with a 170 nm average diameter and a positive potential were obtained. Ethidium bromide was still able to insert into plasmid DNA within the lipoplexes, even though plasmid DNA was demonstrated to be complexed to the lipid by gel electrophoresis. Additionally, DNA was shown to be accessible to DNase I, but significantly resistant to serum enzymatic digestion. Upon intramuscular injection, lipoplexes induced an inhibition of gene expression as compared with naked DNA. Conclusions. The cationic sphingosine/EPC/DNA complexes form weakly compacted structure, potentially labile in vivo, which might be useful for in vivo gene transfer.     

Statistical Modelling of the Formulation Variables in Non-Viral Gene Delivery Systems

Traditionally, optimisation of a gene delivery formulation utilises a study design that involves altering only one formulation variable at any one time whilst keeping the other variables constant. As gene delivery formulations become more complex, e.g. to include multiple cellular and sub-cellular targeting elements, there will be an increasing requirement to generate and analyse data more efficiently and allow examination of the interaction between variables. This study aims to demonstrate the utility of multifactorial design, specifically a Central Composite Design, in modelling the responses size, zeta potential and in vitro transfection efficiency of some prototypic non-viral gene delivery vectors, i.e. cationic liposome-pDNA complexes, and extending the application of the design strategy to more complex vectors, i.e. tri-component lipid:polycation:DNA (LPD). The modelled predictions of how the above responses change as a function of formulation show consistency with an extensive literature base of data obtained using more traditional approaches, and highlight the robustness and utility of the Central Composite Design in examining key formulation variables in non-viral gene delivery systems. The approach should be further developed to maximise the predictive impact of data across the full range of pharmaceutical sciences.     

Intracellular Kinetics of Non-Viral Gene Delivery Using Polyethylenimine Carriers

Purpose Polymeric nucleic acid carriers are designed to overcome one or more barriers to delivery. High molecular weight polyethylenimine (PEI) shows high transfection efficiency but exhibits high cytotoxicity (Fischer et al. Biomaterials, 24:1121–1131 (2003); Peterson et al. Bioconjug. Chem., 13:845–854 (2002)). Nontoxic water-soluble lipopolymer (WSLP) was previously developed using branched poly(ethylenimine) (PEI, mw 1,800) and cholesteryl chloroformate (Han, Mahato, and Kim. Bioconjug. Chem., 12:337–345 (2001)) and is an effective non-viral gene carrier with transfection levels equal or above high molecular weight PEI with a lower cytotoxicity profile. To understand how differences in these polymeric carriers influence transfection, we studied the pharmacokinetics of polymer gene carriers at the cellular level. Materials and Methods Cells were exposed in vitro to different polymeric carriers and the transport of the carriers into different cellular compartments was determined using cellular fractionation and real-time quantitative PCR. A multi-compartment mathematical model was applied to time series measurements of the trafficking of plasmids across each cellular barrier. Results Our result indicates that the chemical modification of WSLP increased the rate parameter for endosomal escape significantly compared to conventional PEI carriers thereby increasing the overall transfection efficiency. Conclusions These results are consistent with the goal of endosomal destabilization of the carrier design. This method provides a quantitative means for assessing different polymer construct designs for gene delivery.     

Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability

Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50–100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.