Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

Development of a Two-Layered Sheet Formulation of 5-Fluorouracil for Application to Rat's Livers to Ensure Controlled Release and Local Drug Disposition.

This study aimed to develop a new and effective application form for the liver surface. We designed a two-layered sheet for the controlled release and local disposition of the anticancer drug, 5-fluorouracil (5-FU), without leakage into the peritoneal cavity. We employed poly(lactic-co-glycolic acid) (PLGA) and hydroxypropyl cellulose (HPC) to form two-layered sheets by attaching a cover sheet and a drug-containing sheet. The prepared two-layered sheets released 5-FU constantly for up to 14 d without any significant leakage from the cover side in vitro. Furthermore, we have applied sheets containing 5-FU to the rat liver surface in vivo. Notably, 5-FU could be detected in the liver attachment region even 28 d after application. The distribution ratio of 5-FU in the attachment region compared to the other liver lobes varied among the sheet formulations with different additive HPC compositions. The area under the liver concentration-time curve (AUC) of 5-FU in the attachment region from 0 to 28 d was the highest in the case of HPC 2% (w/w). This is probably due to the enhanced 5-FU released amount and controlled absorption rate from the liver surface by released HPC. No critical toxic effects were evident by the application of the two-layered sheets from the body weight change and alanine aminotransferase/aspartate aminotransferase (ALT/AST) activities. Consequently, the possible advantage of the two-layered sheets for prolonged retention of a drug in a specific region in the liver was clarified.

Diffusion coefficient of cationic liposomes during lipoplex formation determines transfection efficiency in HepG2 cells.

Cationic lipid-based lipoplexes are well-known for gene delivery. To determine the relationship between physicochemical characteristics and transfection efficiency, cationic liposomes of different sizes were prepared and incubated with plasmid DNA at different temperatures to form lipoplexes. We found that the liposome diffusion coefficient during lipoplex formation strongly correlated with the physicochemical characteristics of lipoplexes, accessibility of plasmid DNA in lipoplexes, and logarithm of gene expression per metabolic activity. Clathrin-mediated endocytosis was the major route for lipoplexes comprising 100 nm-liposomes, as reported previously. As liposome size increased, the major route shifted to lipid raft-mediated endocytosis. In addition, macropinocytosis was observed for all liposome sizes. The role of reactive oxygen species might depend on liposome size and endocytosis. Information from this study would be useful for understanding cationic lipoplex-mediated transfection.

Development of an apolipoprotein E mimetic peptide-lipid conjugate for efficient brain delivery of liposomes.

Liposomes are versatile carriers that can encapsulate various drugs; however, for delivery to the brain, they must be modified with a targeting ligand or other modifications to provide blood-brain barrier (BBB) permeability, while avoiding rapid clearance by reticuloendothelial systems through polyethylene glycol (PEG) modification. BBB-penetrating peptides act as brain-targeting ligands. In this study, to achieve efficient brain delivery of liposomes, we screened the functionality of eight BBB-penetrating peptides reported previously, based on high-throughput quantitative evaluation methods with in vitro BBB permeability evaluation system using Transwell, in situ brain perfusion system, and others. For apolipoprotein E mimetic tandem dimer peptide (ApoEdp), which showed the best brain-targeting and BBB permeability in the comparative evaluation of eight peptides, its lipid conjugate with serine-glycine (SG)5 spacer (ApoEdp-SG-lipid) was newly synthesized and ApoEdp-modified PEGylated liposomes were prepared. ApoEdp-modified PEGylated liposomes were effectively associated with human brain capillary endothelial cells via the ApoEdp sequence and permeated the membrane in an in vitro BBB model. Moreover, ApoEdp-modified PEGylated liposomes accumulated in the brain 3.9-fold higher than PEGylated liposomes in mice. In addition, the ability of ApoEdp-modified PEGylated liposomes to localize beyond the BBB into the brain parenchyma in mice was demonstrated via three-dimensional imaging with tissue clearing. These results suggest that ApoEdp-SG-lipid modification is an effective approach for endowing PEGylated liposomes with the brain-targeting ability and BBB permeability.

Flavonoids Enhance Lipofection Efficiency and Ameliorate Cytotoxicity in Colon26 and HepG2 Cells via Oxidative Stress Regulation.

The generation of reactive oxygen species (ROS) can affect cationic liposome-mediated transfection. In this study, we focused on a specific class of antioxidants, flavonoids, to investigate the transfection efficiency using cationic liposome/plasmid DNA complexes (lipoplexes) in 2D and 3D cultures of Colon26 and HepG2 cells, respectively. All tested flavonoids enhanced the transfection efficiency in 2D Colon26 and HepG2 cells. Among the tested flavonoids, 25 µM quercetin showed the highest promotion effect of 8.4- and 7.6-folds in 2D Colon26 and HepG2 cells, respectively. Transfection was also performed in 3D cultures of Colon26 and HepG2 cells using lipoplexes with quercetin. Quercetin (12.5 µM) showed the highest transfection efficiency at all transfection timings in 3D Colon26 and HepG2 cells with increased cell viability. Flow cytometry revealed that quercetin treatment reduced the population of gene expression-negative cells with high ROS levels and increased the number of gene expression-positive cells with low ROS levels in HepG2 cells. Information from this study can be valuable to develop strategies to promote transfection efficiency and attenuate cytotoxicity using lipoplexes.

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs.

Nucleic acid and genetic medicines are increasingly being developed, owing to their potential to treat a variety of intractable diseases. A comprehensive understanding of the in vivo fate of these agents is vital for the rational design, discovery, and fast and straightforward development of the drugs. In case of intravascular administration of nucleic acids and genetic medicines, interaction with blood components, especially plasma proteins, is unavoidable. However, on the flip side, such interaction can be utilized wisely to manipulate the pharmacokinetics of the agents. In other words, plasma protein binding can help in suppressing the elimination of nucleic acids from the blood stream and deliver naked oligonucleotides and gene carriers into target cells. To control the distribution of these agents in the body, the ligand conjugation method is widely applied. It is also important to understand intracellular localization. In this context, endocytosis pathway, endosomal escape, and nuclear transport should be considered and discussed. Encapsulated nucleic acids and genes must be dissociated from the carriers to exert their activity. In this review, we summarize the in vivo fate of nucleic acid and gene medicines and provide guidelines for the rational design of drugs.

Suppression of Peritoneal Fibrosis by Sonoporation of Hepatocyte Growth Factor Gene-Encoding Plasmid DNA in Mice.

Gene therapy is expected to be used for the treatment of peritoneal fibrosis, which is a serious problem associated with long-term peritoneal dialysis. Hepatocyte growth factor (HGF) is a well-known anti-fibrotic gene. We developed an ultrasound and nanobubble-mediated (sonoporation) gene transfection system, which selectively targets peritoneal tissues. Thus, we attempted to treat peritoneal fibrosis by sonoporation-based human HGF (hHGF) gene transfection in mice. To prepare a model of peritoneal fibrosis, mice were intraperitoneally injected with chlorhexidine digluconate. We evaluated the preventive and curative effects of sonoporation-based hHGF transfection by analyzing the following factors: hydroxyproline level, peritoneum thickness, and the peritoneal equilibration test. The transgene expression characteristics of sonoporation were also evaluated using multicolor deep imaging. In early-stage fibrosis in mice, transgene expression by sonoporation was observed in the submesothelial layer. Sonoporation-based hHGF transfection showed not only a preventive effect but also a curative effect for early-stage peritoneal fibrosis. Sonoporation-based hHGF transfection may be suitable for the treatment of peritoneal fibrosis regarding the transfection characteristics of transgene expression in the peritoneum under fibrosis.

A pH-Adjustable Tissue Clearing Solution That Preserves Lipid Ultrastructures: Suitable Tissue Clearing Method for DDS Evaluation.

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations.

Effect of Chronic Kidney Disease on Hepatic Clearance of Drugs in Rats.

The pharmacokinetics of some hepatically cleared drugs have been reported to fluctuate in patients with renal impairment, but the definitive factors have not been clarified. We compared the pharmacokinetics of some drugs with different hepatic elimination processes in a chronic kidney disease (CKD) rat model, to optimize their administration during kidney injury. We chose indocyanine green (ICG), midazolam (MDZ), and acetaminophen (APAP) as reference drugs to determine changes in hepatic clearance pathways in presence of CKD. Drugs were intravenously administered via the jugular vein to the CKD model rats, previously established by adenine administration, and then, blood, bile, and urine samples were collected. The plasma concentration of ICG, which is eliminated into the bile without biotransformation, increased; and its total body clearance (CLtot) significantly decreased in the CKD group compared to the control group. Moreover, the plasma concentrations of MDZ and APAP, metabolized in the liver by CYP3A and Ugt1a6 enzymes, respectively, were higher in the CKD group than in the control group. The biliary clearances of APAP and its derivative APAP-glucuronide increased in the CKD group, whereas their renal clearances were markedly decreased with respect to those in the control group. Altogether, plasma concentrations of some hepatically eliminated drugs increased in the CKD rat model, but depending on their pharmacokinetic characteristics. This study provides useful information for optimizing the administration of some hepatically cleared drugs in CKD patients.

Co-delivery Systems of Multiple Drugs Using Nanotechnology for Future Cancer Therapy.

Cancer treatments have improved significantly during the last decade but are not yet satisfactory. Combination therapy is often administered to improve efficacy and safety. Drug delivery systems can also improve efficacy and safety. To control the spatiotemporal distribution of drugs, nanotechnology involving liposomes, solid lipid nanoparticles, and polymeric micelles has been developed. Co-delivery systems of multiple drugs are a promising approach to combat cancer. Synergistic effects and reduced side effects are expected from the use of co-delivery systems. In this review, we summarize various co-delivery systems for multiple drugs, including small-molecule drugs, nucleic acids, genes, and proteins. Co-delivery of drugs with different properties is relatively difficult, but some researchers have succeeded in developing such co-delivery systems. Environment-responsive carrier designs can control the release of cargos. Although their preparation is more complicated than that of mono-delivery systems, co-delivery systems can simplify clinical procedures and improve patient QOL.

Interaction of Lipoplex with Albumin Enhances Gene Expression in Hepatitis Mice.

Understanding the in vivo fate of lipoplex, which is composed of cationic liposomes and DNA, is an important issue toward gene therapy. In disease conditions, the fate of lipoplex might change compared with the normal condition. Here, we examined the contribution of interaction with serum components to in vivo transfection using lipoplex in hepatitis mice. Prior to administration, lipoplex was incubated with serum or albumin. In the liver, the interaction with albumin enhanced gene expression in hepatitis mice, while in the lung, the interaction with serum or albumin enhanced it. In normal mice, the interaction with albumin did not enhance hepatic and pulmonary gene expression. Furthermore, hepatic and pulmonary gene expression levels of albumin-interacted lipoplex were correlated with serum transaminases in hepatitis mice. The albumin interaction increased the hepatic accumulation of lipoplex and serum tumor necrosis factor-α level. We suggest that the interaction with albumin enhanced the inflammation level after the administration of lipoplex in hepatitis mice. Consequently, the enhancement of the inflammation level might enhance the gene expression level. Information obtained in the current study will be valuable toward future clinical application of the lipoplex.

Influence of Liver Intoxication by Carbon Tetrachloride or D-Galactosamine on Absorption of Fluorescein Isothiocyanate-Dextran-10 and Other Marker Compounds with Different Molecular Weights from the Rat Liver Surface.

We examined the influence of liver disease on the absorption from the liver surface of fluorescein isothiocyanate (FITC)-dextran 10 (FD-10, MW: 11000) and several marker compounds with different molecular weights. The purpose of this study was to determine the feasibility of liver surface application of macromolecular compounds in the disease state. We used male Wistar rats treated with carbon tetrachloride (CCl4) or D-galactosamine (GAL). FD-10 and other marker compounds were applied to the liver surface using a cylindrical diffusion cell in liver-intoxicated rats. The blood, bile, urine, and the remaining solution in the diffusion cell were collected for assay. FD-10 was absorbed by first-order kinetics from the liver surface in the liver-intoxicated rat models. The calculated rate constant ka values in the normal, CCl4 and GAL groups were 0.000965, 0.00125 and 0.00104 min-1, respectively. Increased absorption of FITC-dextrans in the liver-intoxicated rats was observed. In both CCl4 and GAL groups, an inverse relationship was observed between the molecular weight and ka from the rat liver surface of the marker compounds. The limits of the molecular weight absorbed from the liver surface were extrapolated to be 71200, 135000, and 105000 in the normal, CCl4, and GAL groups, respectively. In conclusion, increased absorbability from the rat liver surface indicates that liver surface application for liver targeting of macromolecules in the diseased state is indeed feasible. Therefore, our findings can support further research on liver surface application of drugs under liver disease.

Tissue suction-mediated gene transfer to the beating heart in mice.

We previously developed an in vivo site-specific transfection method using a suction device in mice; namely, a tissue suction-mediated transfection method (tissue suction method). The aim of this study was to apply the tissue suction method for cardiac gene transfer. Naked plasmid DNA (pDNA) was intravenously injected in mice, followed by direct suction on the beating heart by using a suction device made of polydimethylsiloxane. We first examined the effects of suction conditions on transgene expression and toxicity. Subsequently, we analyzed transgene-expressing cells and the transfected region of the heart. We found that heart suction induced transgene expression, and that -75 kPa and -90 kPa of suction achieved high transgene expression. In addition, the inner diameter of the suction device was correlated with transgene expression, but the pressure hold time did not change transgene expression. Although the tissue suction method at -75 kPa induced a transient increase in the serum cardiac toxicity markers at 6 h after transfection, these markers returned to normal at 24 h. The cardiac damage was also analyzed through the measurement of hypertrophic gene expression, but no significant differences were found. In addition, the cardiac function monitored by echocardiography remained normal at 11 days after transfection. Immunohistochemical analysis revealed that CD31-positive endothelial cells co-expressed the ZsGreen1-N1 reporter gene. In conclusion, the tissue suction method can achieve an efficient and safe gene transfer to the beating heart in mice.

Evaluation of mRNA expression of drug-metabolizing enzymes in acetaminophen-induced hepatotoxicity using a three-dimensional hepatocyte culture system.

1. The expression and activity of drug-metabolizing enzymes are known to affect the pharmacokinetics of drugs metabolized in the liver. Here, we assessed the effect of acetaminophen (APAP)-induced hepatotoxicity on the mRNA expression of drug-metabolizing enzymes and elucidated the underlying mechanism using three-dimensional (3D) cultures of HepG2 cells.2. 3D culture cells enabled us to establish an in vitro model of APAP-induced hepatotoxicity which showed the increase in N-acetyl-p-benzoquinone imine production, reactive oxygen species (ROS) generation and cellular injury.3. In this 3D culture model, APAP treatment significantly increased the mRNA expression of drug-metabolizing enzymes (cytochrome P450 [CYP]3A4, CYP2E1 and UDP-glucuronosyltransferase 1A6) and their nuclear receptors (pregnane X receptor and constitutive androstane receptor) compared with untreated cells. Treatment with N-acetylcysteine, a therapeutic agent for APAP-induced hepatotoxicity, suppressed these increases. In addition, the mRNA expression of drug-metabolizing enzymes and nuclear receptors were elevated depending on the concentration of H2O2, one of ROS involved in the development of APAP-induced hepatotoxicity. The mRNA expression of nuclear receptors increased before that of drug-metabolizing enzymes.4. In conclusion, ROS may induce the mRNA expression of nuclear receptors and promote the transcription of drug-metabolizing enzymes in the in vitro model of APAP-induced hepatotoxicity.

Monitoring method for transgene expression in target tissue by blood sampling.

In this study, we have developed a novel method to monitor transgene expression in tissues by blood sampling. We administered plasmid DNA (pDNA) encoding non-secretory form of firefly luciferase as a reporter gene and pDNA encoding secretable Gaussia princeps luciferase as a monitor gene simultaneously into mice. Good positive correlations were found between log-transgene expression of the reporter gene and the monitor gene in the treated muscle, between the monitor gene in the treated muscle and plasma, and consequently between the reporter gene in the treated muscle and the monitor gene in plasma after naked pDNA transfer into the muscle of mice. Such positive correlations were also found with gastric serosal surface instillation of naked pDNA, intravenous injection of lipoplex, and hydrodynamics-based injection of naked pDNA. We developed monitoring method of transgene expression in tissues by blood sampling, which was named 'Therapeutic transgene monitoring (TTM)', after 'Therapeutic drug monitoring (TDM)'.

Effect of renal ischaemia/reperfusion-induced acute kidney injury on pharmacokinetics of midazolam in rats.

OBJECTIVES: This study aimed to investigate the effects of renal ischaemia/reperfusion (I/R)-induced acute kidney injury (AKI) on the distribution of midazolam (MDZ), a probe drug for cytochrome P450 3A (CYP3A) activity. METHODS: We established an AKI model inducing ischaemia of both renal pedicles for 60 min followed by 24-h reperfusion. MDZ was administered intravenously (i.v.) to the rats via the jugular vein, and then, blood samples were collected to determine the plasma concentration of MDZ. KEY FINDINGS: While the plasma concentration of MDZ after i.v. administration was decreased in the I/R rats, the tissue concentration was not altered. In addition, the tissue-to-plasma (T/P) ratio of MDZ was increased in the I/R rats. The unbound fraction of MDZ and the level of indoxyl sulphate (IS) in plasma were elevated in the I/R rats. Furthermore, the unbound fraction of MDZ was significantly increased by the addition of IS. CONCLUSIONS: These results indicated that the displacement of albumin-bound MDZ by IS changed the unbound fraction of MDZ and elevated the T/P ratio of MDZ in I/R rats.

Application of Direct Sonoporation from a Defined Surface Area of the Peritoneum: Evaluation of Transfection Characteristics in Mice.

In the present study, we developed a sonoporation system, namely "direct sonoporation", for transfecting the peritoneum from a defined surface area to avoid systematic side effects. Here, the transfection characteristics are explained because there is less information about direct sonoporation. Naked pDNA and nanobubbles were administered to diffusion cell attached to the visceral and parietal peritoneum from the liver and peritoneal wall surface, respectively. Then, ultrasound was irradiated. Direct sonoporation showed a higher transfection efficacy at the applied peritoneum site from the liver surface while other sites were not detected. Moreover, transgene expression was observed in the peritoneal mesothelial cells (PMCs) at the applied peritoneum site. No abnormality was observed in the inner part of the liver. Although transgene expression of the visceral peritoneum was tenfold higher than that of the parietal peritoneum, transgene expression was observed in the PMCs on both the applied peritoneum sites. These results suggest that direct sonoporation is a site-specific transfection method of the PMCs on the applied peritoneum site without transgene expression at other sites and show little toxicity in the inner tissues at the applied site via cavitation energy. This information is valuable for the development of an intraperitoneal sonoporation device for treatment of peritoneal diseases such as peritoneal fibrosis.

Targeted co-delivery of protein and drug to a tumor in vivo by sophisticated RGD-modified lipid-calcium carbonate nanoparticles.

Synchronized bio-distribution of combination therapies has several merits such as synergistic effects and reduced side-effects. Co-delivery of a protein and small molecule drug using a single nanocarrier is challenging because they possess totally different characteristics. Herein, we report the development of sophisticated nanoparticles composed of lipids, calcium carbonate and RGD peptide ligands for the co-delivery of a protein and small molecule drug combination via a simple preparation method. A 'one-step' ethanol injection method was employed to prepare the highly organized nanoparticles. The nanoparticles exhibited a spherical shape with ca. 130 nm diameter, and clearly had an integrated lipid layer covering the periphery. As a ligand, an RGD-modified lipid was post-inserted into the nanoparticles, which was important to overcome the 'PEG dilemma'. The pH-sensitivity of the targeted nanoparticles contributed to the efficient intracellular co-delivery of a protein and drug combination in Colon26 tumor cells, and noticeably improved their accumulation in the tumor region of xenograft mice. Synchronized bio-distribution of the protein and drug was achieved, which was the foundation for the synergistic effects of the combination. The targeting capability of the nanoparticles along with their pH-sensitive drug release and the synchronized bio-distribution of their cargos led to the significant antitumor activity of the SOD and paclitaxel combination in mice. This study provides novel information for the design and preparation of functionalized nanoparticles for the delivery of a protein/drug combination in vivo.

Ultrasound-responsive nanobubble-mediated gene transfection in the cerebroventricular region by intracerebroventricular administration in mice.

AIM: Intracerebroventricular (ICV) administration of ultrasound-responsive bubbles and cranial ultrasound irradiation is reported as a transfection system for the cerebroventricular region. This study aimed to characterize the transfection system with respect to transfection efficiency, spatial distribution of transgene expression, and safety. METHODS: Plasmid DNA was transfected to mouse brain by ICV injection of ultrasound-responsive nanobubbles, followed by ultrasound irradiation to brain. Spatial distribution of transgene expression in the cerebroventricular region was investigated using multicolor deep imaging. RESULT: This transfection system efficiently transferred the transgene to the choroid plexus with no morphological change or cerebral hemorrhage. Moreover, sustained secretion of transgenic protein was achieved by transferring the transgene encoding the secretable protein. CONCLUSION: We successfully developed an ultrasound-responsive nanobubbles-mediated method for gene transfection into the cerebroventricular region via ICV administration in mice.

Edaravone, a cytoprotective drug, enhances transgene expression mediated by lipoplexes in HepG2 cells and mice.

A requirement of gene therapy is efficient nucleic acid delivery. However, the application of cationic liposomes to gene therapy is restricted by their inefficient transfection capacity, which may be caused by cytotoxicity. This cytotoxicity is highly dependent on cationic lipid-induced reactive oxygen species (ROS). Here, to provide cellular protection, we used edaravone, an efficacious anti-oxidative drug, to scavenge ROS during transfection using cationic liposome/plasmid DNA complexes (lipoplexes). Both free edaravone and edaravone-loaded liposomes (EDLPs) enhanced transgene expression in the human hepatoma cell line, HepG2, while EDLPs decreased the effective dose of edaravone. The cellular protective effect of edaravone was found to decrease the cytotoxicity of cationic liposomes. Edaravone was also effective in the commercial product, Lipofectamine® 3000, which may expand the application of edaravone to promote transfection efficiency. Compared with free edaravone, EDLPs also showed superior transgene expression in mice. Our findings will promote the development of efficient and safe gene therapy.