Evaluation of proinflammatory cytokine production and liver injury induced by plasmid DNA/cationic liposome complexes with various mixing ratios in mice

The purpose of this study was to investigate the cytokine production and liver injury induced by lipoplexes prepared with DOTMA/cholesterol and DOTAP/cholesterol liposomes with various mixing ratios in mice. Lipoplexes were prepared with pCMV-Luc and DOTMA/cholesterol or DOTAP/cholesterol liposomes. After intravenous administration into the mice, organ luciferase activity and serum TNFα and ALT were measured to evaluate the transfection efficacy, cytokine production and liver injury. After intravenous administration of these lipoplexes, basically the serum TNFα and ALT levels were in agreement with the transfection efficacy of the lipoplexes. The cytokine production and liver injury were markedly suppressed by reducing the pDNA dose, and achieved normal levels at a pDNA dose of 0.47 mg/kg. As far as the effects of the charge ratio at this low pDNA dose are concerned, the charge ratios of the lipoplexes affected the transfection efficacy, but not the cytokine production and liver injury. After intravenous administration of either DOTAP/cholesterol or DOTMA/cholesterol liposomes, serum TNFα and ALT levels were normal, suggesting that liver injury as well as cytokine production was caused by lipoplexes, but not by cationic liposomes. This information will be valuable for the future optimization of the preparation conditions of lipoplexes for use in clinical gene therapy.     

Poly(L-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 approximately 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Poly(l-lactide-co-glycolide) nanospheres conjugated with a nuclear localization signal for delivery of plasmid DNA

Polymeric nanospheres fabricated from biodegradable poly(lactide-co-glycolide) (PLGA) have been extensively investigated for applications in gene delivery. In this study, we show that the covalent conjugation of a nuclear localization signal (NLS, SV40 peptide) on PLGA nanospheres enhances the gene transfection efficiency. NLS conjugated PLGA copolymer was prepared by using a coupling reaction between maleimide-terminated PLGA copolymer and NLS in the presence of Imject maleimide conjugation buffer. PLGA nanospheres encapsulating plasmid (pDNA) were prepared by using a double emulsion-solvent evaporation method. The kinetics of in vitro release of pDNA from PLGA nanospheres was determined with UV in phosphate buffered saline (PBS). Gene transfection efficiency in human dermal fibroblasts was tested in vitro using nanospheres encapsulating the luciferase gene. The conjugation of the NLS peptide to the PLGA nanospheres could improve the nuclear localization and/or cellular uptake of PLGA nanosphere/pDNA constructs and thereby improve the transfection efficiency of a PLGA nanosphere gene delivery system. The pDNA was released from PLGA nanospheres over nine days. NLS conjugation enhanced the gene transfection efficiency in vitro by 1.2 ～ 3.2-fold over 13 days. PLGA/pDNA nanospheres appeared to be superior to PEI/pDNA complexes for the long-term expression of pDNA. Furthermore, the level of the sustained gene expression of the PLGA nanospheres was enhanced by the conjugation of NLS to the PLGA nanospheres. This study showed that the NLS conjugation enhanced the gene transfection efficiency of the PLGA nanosphere gene delivery system in vitro and that the enhanced gene expression was sustained for at least 13 days.     

Preparation and characterization of magnetic cationic liposome in gene delivery

Low transfection efficiency in vivo and failure to deliver therapeutic nucleic acids to the target organs limit the use of cationic liposomes (CLs) in gene therapy. Magnetic drug targeting (MDT) was applied in this study to improve the transfection efficiency and overcome the limitations. Magnetic cationic liposomes (MCLs) were prepared by incorporating MAG-T (magnetite) into CLs. The inclusion of relatively high concentration of MAG-T significantly increased the size of liposomes/lipoplexes, reduced the zeta potential, and decreased the cell viability. The transfection efficiency of MCLs in gene delivery was evaluated by using plasmid DNA (pDNA) containing a luciferase reporter gene in THLE-3 cells. Results suggested that the transfection efficiency of MCLs/pDNA complexes with a relatively lower content of MAG-T (0.75 mg/ml) was the same as that of CLs/pDNA complexes without a magnetic field but was higher (about 2.6-fold) with magnetic induction. Finally, using MCLs/pDNA complexes and a static magnetic field placed over the liver of rats, luciferase reporter gene expression in the liver increased as compared to MCLs/pDNA complexes and CLs/pDNA complexes in the absence of an external magnetic field.     

Hydration of surfactant-modified and PEGylated cationic cholesterol-based liposomes and corresponding lipoplexes by monitoring a fluorescent probe and the dielectric relaxation time

For the optimization of plasmid DNA (pDNA)-cationic lipid complexes and lipoplex delivery, proper indexes of the physicochemical properties of lipoplexes are required. In general, the characteristics of lipoplexes are defined by particle size and zeta-potential at various mixing ratios of cationic liposomes and pDNA. In this study, we characterized the hydration level of surfactant-modified and PEGylated cationic cholesterol-based (OH-Chol) liposomes and their lipoplexes by monitoring both the fluorescent probe laurdan and the dielectric relaxation time. Fluorescence measurement using laurdan detected hydration of the headgroup of lipids in surfactant-modified liposomes and PEGylated DOTAP-liposomes, but hardly any fluorescence was detected in PEGylated OH-Chol-liposomes because the PEG layers may extend and cover the fluorescent maker. On the other hand, the measurement of dielectric relaxation time of water molecules revealed total hydration, including hydration of the PEG layer and the headgroup of cationic lipids. Furthermore, we found an inverse correlation between hydration level and cellular uptake of PEGylated lipoplexes (R=0.946). This finding indicated that the dielectric relaxation time of water molecules provides an important indicator of hydration of liposome and lipoplexes along with the fluorescence intensity of laurdan.     

Stearylated INF7 peptide enhances endosomal escape and gene expression of PEGylated nanoparticles both in vitro and in vivo

We previously reported on a stearylated INF7 peptide (str-INF7), which enhances the endosomal escape of an octaarginine (R8)-modified liposomal particle encapsulating plasmid DNA (pDNA) in a fusion-independent manner. This study examined whether this peptide derivative enhanced the endosomal escape and gene expression of PEGylated liposomes encapsulating pDNA. We used a PEGylated, R8-modified multifunctional envelope-type nanodevice (R8-MEND) as a model for PEGylated liposomes. Polyethylene glycol 2000 (PEG2000) attached to two different anchors, distearoylphosphatidylethanolamine (DSPE-PEG) or dimyristoylphosphatidylethanolamine (DMPE-PEG), was used to modify the R8-MEND in the presence or absence of two different concentrations of str-INF7. Modification of the PEGylated R8-MEND with str-INF7 resulted in luciferase gene expression levels in HeLa cells that were 73-fold and 24-fold higher than the corresponding value for an unmodified MEND in the case of DSPE-PEG and DMPE-PEG, respectively. The endosomal escape of the PEGylated R8-MEND was improved by str-INF7, as confirmed by confocal laser scanning microscopy. Furthermore, modification with str-INF7 enhanced the hepatic gene expression of the R8-MEND modified with DSPE-PEG and DMPE-PEG by 95-fold and 1885-fold, respectively, after intravenous injection in mice. Collectively, these data demonstrate that str-INF7 can be a useful device for enhancing the endosomal escape even for PEGylated liposomes encapsulating pDNA.     

In vivo studies of dialkynoyl analogues of DOTAP demonstrate improved gene transfer efficiency of cationic liposomes in mouse lung

A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure-activity studies demonstrate that replacement of the cis-double bonds of DOTAP with triple bonds in varying positions alters both the physical properties of the resultant cationic liposome-DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

Non-linear pharmacodynamics in the transfection efficiency of a non-viral gene delivery system

Transfection efficiencies using LipofectAMINE varied by more than three orders of magnitude depending on the concentrations of lipid and plasmid DNA (pDNA) used to prepare the lipoplexes. When lipoplexes were formed at lower concentrations a striking positive but non-linear relationship was found between dose and transfection efficiency, while at higher (i.e., normally used) concentrations a linear relationship was maintained. To determine the contribution of intracellular pharmacokinetics (PK) and pharmacodynamics (PD) to the observed nonlinearity, we quantified pDNA in whole cells and nuclei by real-time PCR and compared the results with the transfection efficiencies. There was no significant difference in the efficiency of intracellular PK; however, a remarkable difference was observed in the efficiency of PD. Analysis of individual cells by confocal laser scanning microscopy (CLSM) revealed that the amount of nuclear-delivered pDNA was higher for lipoplexes prepared at the normal concentration (NCL) compared to those of lipoplexes prepared at low concentration (LCL). Moreover, the size of the NCL was larger than that of the LCL. Both the size of the lipoplex particle and the dose appear to contribute to the non-linear efficiency of PD. These results emphasize the need to control not only intracellular PK, but also PD for the rational development of non-viral gene delivery systems.     

Nonviral approach for targeted nucleic acid delivery

Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.     

Cellular association and cytotoxicity of doxorubicin-loaded immunoliposomes targeted via Fab' fragments of an anti-CD74 antibody

The purpose of our research was to evaluate in vitro therapeutic efficacy of doxorubicin (DXR)-loaded immunoliposomes with Fab' fragments of the anti-CD74 antibody LL1 attached to the surface. LL1 is well suited for targeting purposes because it is internalized very fast by B-lymphoma cells. However, at in vivo application whole antibodies show fast clearance in circulation. Taking this fact into consideration, this study was initiated to elucidate the prospects of using Fab' fragments of LL1 in stead of the whole antibody for future targeting in vivo of DXR-loaded liposomes. The Fab' fragments were covalently attached to the surface of sterically stabilized liposomes by use of a PEG-based heterobifunctinal coupling agent. LL1 Fab' conjugated sterically stabilized DXR liposomes showed approximately six times faster accumulation of the drug in Raji human B-lymphoma cells than nontargeted liposomes. In vitro cytotoxicity, quantitated by a tetrazolium assay, against Raji cells gave IC(50) values of 0.13, 0.45, and 0.11 microM for DXR-loaded immunoliposomes, DXR-loaded liposomes and free drug, respectively. The results from this study suggest that DXR-loaded immunoliposomes targeted with Fab' fragments from the anti-CD74 antibody LL1 could be a useful system for future in vivo experiments.     

壳聚糖纳米粒用作基因递送载体的初步研究

目的初步研究基因壳聚糖纳米粒的性质和转染活性。方法用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;用荧光分光光度法测定基因包封率;用凝胶阻滞分析和荧光扫描测定基因在纳米粒中的位置;用体外基因转染实验定性评价纳米粒的转染活性。结果纳米粒形态多呈球形,平均粒径为218.9 nm,多分散度为0.276,zeta电位为+21.2 mV;基因包封率为99.6%;凝胶阻滞分析和荧光扫描表明基因几乎全部被包裹在纳米粒内部,表面吸附很少;体外基因转染实验表明基因壳聚糖纳米粒能够转染人胚胎肾细胞(HEK293)和肝癌细胞(HepG2),基因能够在这两种细胞中表达。结论壳聚糖纳米粒能将基因递送到细胞内并且基因能够表达,因此可以用作基因药物载体。     

Effect of oligopeptides on gene expression: comparison of DNA/peptide and DNA/peptide/liposome complexes

Plasmid DNA is known to form complexes with a variety of cationic peptides and lipids, which have been explored as possible carriers for DNA transfection in mammalian cells. We synthesized oligopeptides consisting of nine amino acid residues including lysine (K), tryptophan (W), and cysteine (C), and also their symmetrical dimmers with a disulfide bond as possible carriers. The pDNA(pGL3)/oligopeptide complexes generally showed poor transfection efficiencies but little cytotoxicity for HeLa S3. The ternary system of pDNA/oligopeptide/liposome containing cationic liposomes formulated from the cholesterol derivative (DMB-Chol) and dioleoylphosphatidylethanolamine (DOPE) showed 10(4)-10(5)-fold greater effective gene expression (10(8)-10(9) level, RLU/min/mg protein) than those of the corresponding pDNA/oligopeptide complexes. In the presence of 10% serum, the ternary complexes were maintained at 10(7) levels. The ethidium bromide exclusion studies showed the ternary complexes have much greater affinity to pDNA than the corresponding pDNA/oligopeptide complexes. Plasmid sensitivity against DNase I degradation showed that the ternary complexes were well protected from the digestion. Synthetic oligopeptides are active as potential enhancers for DOPE-containing cationic liposome-mediated transfection. These findings have implications for successful in vivo transfection.     

Peptide-mediated lipofection is governed by lipoplex physical properties and the density of surface-displayed amines

Peptides can potentiate lipid-mediated gene delivery by modifying lipoplex physiochemical properties to overcome rate-limiting steps to gene transfer. The objectives of this study were to determine the regimes over which cationic peptides enhance lipofection and to investigate the mechanism of action, such as increased cellular association resulting from changes in lipoplex physical properties. Short, cationic peptides were incorporated into lipoplexes by mixing peptide, lipid and DNA. Lipoplexes were characterized using gel retardation, dynamic light scattering, and fluorescent microscopy, and the amount of surface-displayed amines was quantified by fluorescamine. Size, zeta potential, and surface amines for lipoplexes were dependent on peptide/DNA ratio. Inclusion of peptides in lipoplexes resulted in up to a 13-fold increase in percentage of cells transfected, and up to a 76-fold increase in protein expression. This transfection enhancement corresponded to a small particle diameter and positive zeta potential of lipoplexes, as well as increased amount of surface-displayed amines. Relative to lipid alone, these properties of the peptide-modified lipoplexes enhanced cellular association, which has been reported as a rate-limiting step for transfection with lipoplexes. The addition of peptides is a simple method of lipofection enhancement, as direct chemical modification of lipids is not necessary for increased transfection.     

阴离子脂质体－阳离子脂质体复合物介导的基因转染

目的评价阴离子脂质体-阳离子脂质体复合物介导质粒转移至HepG2肝癌细胞中及其毒副作用。方法制备携载表达绿色荧光蛋白质粒的阳离子脂质体,与阴离子脂质体形成复合物。测定脂质体复合物的zeta电位,凝胶阻滞实验考察质粒包封情况,流式细胞仪测量各阴离子脂质体-阳离子脂质体复合物的转染效率,MTT法检测细胞毒性。结果复合物能完全包裹质粒,其zeta电位低于阳离子脂质体zeta电位;脂质体复合物介导的转染效率略低于阳离子脂质体,其细胞生存率高于阳离子脂质体。结论阴离子脂质体-阳离子脂质体复合物在降低细胞毒性的同时,可实现对HepG2细胞较高的转染效率。     

Insights into the mechanism of magnetofection using MNPs-PEI/pDNA/free PEI magnetofectins

Magnetofection is an efficient new physical gene transfection technology. Despite its effective gene delivery capability, till now relatively little work has been conducted on the mechanism of magnetofection, especially the intracellular fates of the components of magnetofectins and their effects on magnetofection. In this study, we investigated the mechanism of magnetofection using magnetofectins that were prepared via electrostatic self-assembly of the three components: polyethyleneimine (PEI)-coated magnetic nanoparticles (MNPs-PEI), plasmid DNA (pDNA) and PEI in the free form (free PEI). TEM observation and agarose gel electrophoresis assays have indicated MNPs play the role of driving magnetofectins to the cell surface without entering into the nucleus. Confocal microscopic tracking of fluorescence-labeled PEI has shown that the free PEI (green) can be found in the nucleus but almost all of the MNPs-PEI (red) are confined in the cytoplasm in COS-7 cells 30 min post-transfection or in SPC-A1 cells 90 min post-transfection, implying that the pDNA/PEI complex must separate from MNPs-PEI before entering into the nucleus. In addition, reporter gene assays showed the magnetofectins, in which the free PEI was absent, failed to transfect SPC-A1 or COS-7 cell lines; and there was an optimal ratio of the constituents of magnectofectins to achieve optimal transfection efficiency by balancing stable complex formation and facile release of PEI/pDNA from the complex. In summary, our findings further the knowledge of magnetofection and can be helpful for the design and preparation of gene delivery vehicles for effective magnetofection.     

Single-cell mRNA transfection studies: Delivery,kinetics and statistics by numbers

In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose–response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs.From the Clinical EditorThis team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules.     

Enhancement of gene transfection into human dendritic cells using cationic PLGA nanospheres with a synthesized nuclear localization signal

Effective delivery of DNA encoding antigen into the dendritic cells (DCs), which are non-dividing cells, is very important for the development of DNA vaccines. In a previous study, we developed the PLGA nanospheres that contained a cationic nanomaterial and showed high transfection efficiency in COS7 cells, which divide. In the present study, to produce an effective vector for the DNA vaccines, the gene expression and intracellular trafficking of pDNA complexed with PLGA/PEI nanospheres, in combination with an NF-κB analog as a nuclear localization signal (NLS) and electroporation were evaluated in human monocyte-derived DCs (hMoDCs). Cellular uptake of pDNA both in COS7 cells and hMoDCs was enhanced using the PLGA/PEI nanospheres. On the other hand, the PLGA/PEI nanospheres significantly promoted the transfection in COS7 cells, but had almost no effect on transfection in hMoDCs. The intranuclear transport of pDNA by PLGA/PEI nanospheres in COS7 cells was significantly higher than that in hMoDCs. These results indicate that pDNA complexed with PLGA/PEI nanospheres cannot enter into the nuclei of non-dividing cells. However, PLGA/PEI nanospheres combinated with NLS and electroporation (experimental permeation enhancer) greatly elevated the transfection efficiency by improvement of not only intracellular uptake but also intranuclear transport of pDNA in the hMoDCs. Thus, this delivery system using nanospheres combined with synthesized NLS might be applicable to DC-based gene vaccines when much non-invasive application such as needle-free injector should be required.