Efficient Packaging of Plasmid DNA Using a pH Sensitive Cationic Lipid for Delivery to Hepatocytes

Abstract

Plasmid DNA (pDNA) is expected to be a new class of medicine for treating currently incurable diseases. To deliver these nucleic acids, we developed a liposomal delivery system we have called a multifunctional envelope-type nano device (MEND). In this report, we demonstrate that a MEND containing a pH-sensitive cationic lipid, YSK05 (YSK-MEND), efficiently delivered pDNA via systemic injection, and that its expression was highly dependent on the encapsulation state of the pDNA. In the preparation, the pH, ionic strength, and sodium chloride (NaCl) concentration of the lipid/pDNA mixture strongly affected the encapsulation efficiency of pDNA. Additionally, the transgene expression of luciferase in the liver by the injected YSK-MEND was dependent on the encapsulation state of pDNA rather than the nature of the YSK-MEND. Confocal laser scanning microscopy findings revealed that injection of the YSK-MEND led to homogenous gene expression in the liver compared to injection via the hydrodynamic tail vein (HTV). Concerning the safety of the YSK-MEND, a transient increase in the activity of liver enzymes was observed. However, no significant adverse events were observed. Taken together, the YSK-MEND represents a potentially attractive therapy for the treatment of various hepatic diseases.

Gene delivery undoubtedly holds great potential for the treatment of hereditary diseases. Actually, an adeno-associated viral vector coding lipoprotein lipase was approved for the treatment of familial hyperchylomicronemia in Europe in 2012. Despite the advantages of gene delivery therapy, recent advances in gene delivery, including plasmid DNA (pDNA), have been relatively restricted compared to the delivery of artificially synthesized oligonucleotides, including small interfering RNA (siRNA), anti-sense oligonucleotides and anti-micro RNA oligonucleotides because of difficulty associated with developing a safe, effective non-viral vector for pDNA.¹⁾

Hydrodynamic tail vein (HTV) injection is an established method for the successful gene delivery to hepatocytes,²⁾ myocytes,³⁾ mitochondria of limb muscles⁴⁾ and so on. Jacobs et al. reported that transgene expression in the liver as the result of HTV injection equaled that of adenovirus injection.⁵⁾ Due to its high potency, HTV injection is currently under preclinical evaluation.⁶⁾ In an attempt to achieve successful carrier-mediated pDNA delivery, we developed a liposomal delivery system, a multi functional envelope-type nano device (MEND) for delivering siRNA, we recently designed a pH-sensitive cationic lipid, YSK05, which consists of tertiary amine head as a hydrophilic moiety and an octadeca-9,12-dien chain as a hydrophobic moiety.⁷⁾ The YSK-MEND effectively delivered siRNA to the liver,⁸⁾ tumors⁹⁾ and tumor endothelial cells.¹⁰⁾ This type of YSK-MEND would also be predicted to have potential for pDNA delivery.

The goal of this research was to establish a method for encapsulating pDNA within liposomal carriers using YSK05 and to achieve an effective transgene expression in the liver when the pDNA carriers are intravenously injected. The findings reported herein show that the pH-sensitive YSK-MEND nano carrier loaded with pDNA exhibited a high gene expression.

MATERIALS AND METHODS

Materials

Polyethyleneglycol 1,2-dimyristoyl-rac-glycerol (PEG-DMG) was purchased from the NOF CORPORATION (Tokyo, Japan). YSK05 was synthesized as previously reported.⁷⁾ Cholesterol (chol) was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Luciferase assay systems was obtained from Promega (Madison, WI, U.S.A.). 1,2-Dipalmitoyl-sn-glycerophosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) and 1,2-dioloyl-3-trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL, U.S.A.). CpG free pDNAs coding luciferase and mCherry were constructed as previously reported.¹¹⁾

Preparation of the MEND

The R8-GALA-MEND was prepared as previously reported.¹²⁾ The YSK-MEND was constructed by a slightly modified tertiary-butanol (t-BuOH) dilution method for siRNA delivery.¹³⁾ Briefly, pDNA (20–160 µg) in citrate buffer (134 µL, 2 mM, pH 4.0–7.0) was gradually added to the lipid solution in 90% t-BuOH (466 µL, YSK05/chol 70/30 or YSK05/helper lipid/chol 60/10/30, total 3000 nmol, PEG-DMG 3 mol% of total lipid). For the efficient encapsulation of pDNA, sodium chloride (NaCl) was added to the citrate buffer at concentrations of 0–500 mM. The mixture was then added to phosphate buffered saline (PBS) (2.0 mL, pH 7.4) via a 25 G syringe with vigorous stirring, and PBS (6.0 mL) was added to the solution to dilute the concentration of t-BuOH. The resulting mixture was ultracentrifuged using a Vivaspin (Sartorius, Göttingen, German) twice to remove residual t-BuOH (1000×g, 20 min, room temperature). The particle size distribution and z-potential of the YSK-MENDs were determined by a Zetasizer Nano ZS ZEN3600 instrument (Malvern Instruments, Worchestershire, U.K.). Encapsulation efficiency (EE) was measured by agarose gel electrophoresis (AGE) and PicoGreen (Invitrogen, Carlsbad, CA, U.S.A.). An aliquot of the YSK-MEND (20 ng) was mixed with (A) distilled deionized water (DDW) or (B) polyasparate (pAsp; 0.83 mg/mL) or (C) polyasparate (0.83 mg/mL) and sodium dodecylsulfate (0.83% (w/v)), and then subjected to 1% AGE in tris-borate buffer. The pDNA was stained in etidium bromide (1 µg/mL), and visualized with a Dolphin-View2 (KURABO INDUSTRIES, Okayama, Japan). The EE was calculated from the fluorescence intensity (FI) obtained by ImageJ software using a following equation; EE (%)=(FI of sample (C)−FI of sample (B))/FI of sample (C). Nitrogen/Phosphate (N/P) ratio was calculated as below;   

Animal Study

Male, 4-week-old ICR mice were purchased from SLC (Shizuoka, Japan). Under diethylether anesthesia, the YSK-MEND was injected via the tail vein at the indicated doses. Alanine aminotransferase (ALT) and asparagine transaminase (AST) were used as a hepatological toxicity indicator using a CII-Test Wako Kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) according to the manufacturer’s protocol. To assess the in vivo transgene expression of MENDs, ICR mice were injected with the YSK-MEND at the indicated doses, which were adjusted by the pDNA EE of each carrier. Dissected liver tissue was homogenized by PreCellys (Bertin Technologies, Montigny-Le-Bretonneux, France) in 1× Passive Lysis Buffer (Promega, Madison, WI, U.S.A.). After centrifugation (20000×g, 10 min, 4°C), the luminescence of aliquots of the supernatant (20 µL) was measured by a luminometer (Luminescencer-PSN; ATTO Corporation, Tokyo, Japan). The luminescence was normalized to the protein concentration of the homogenates, which was determined by means of a bicinchoninic acid (BCA) assay. The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the Guide for the Care and Use of Laboratory Animals. For an evaluation of a hepatological toxicity, AST and ALT in serum were determined by CII-Test Wako Kit (Wako Pure Chemical Industries, Ltd.)

Con-Focal Laser Scanning Microscopy Observations

Liver tissue collected from deeply anesthetized mice was embedded in OCT compound and sectioned by a CM-3050S (Leica, Wetzlar, Germany) in 15 µm thickness. Nuclei were stained by dipping the slide glass in 10 µg/mL of a Hoechst33342 solution for 10 min. The slices were observed with a FV10i-LIV Microscope (Olympus Corporation, Tokyo, Japan).

Statistical Analysis

Comparisons between multiple treatments were performed using one way ANOVA, followed by the Bonferroni test or SNK test. Pair-wise comparisons between treatments were made using a Student’s t-test. A p-value of <0.05 was considered to be significant.

RESULTS

Gene Expression in the Liver Tissue

To assess the transfection efficiency of the YSK-MEND in the liver, ICR mice were administered with the pDNA encoding luciferase encapsulated in the YSK-MEND. The pDNA was formulated into YSK-MEND using the same method as was used in the case of siRNA. Hydrodynamic tail vein (HTV) injection, which is known to achieve a very high transgene expression in the liver was regarded as a positive control. While high gene expression was observed in a group of mice treated with HTV injection, the YSK-MEND prepared with the original protocol showed a dramatically low luciferase expression (Fig. 1).

Fig. 1. Transgene Expression of the pDNA-Encapsulating YSK-MEND Prepared Using the Same Method as for siRNA

Luciferase expression in the liver was determined by means of a luminometer after an injection of 0.5 µg/mouse by hydrodynamics tail vein injection (HTV) injection with a YSK-MEND dose of 10 µg/mouse. Data represents mean and standard deviation. ** p<0.01 Student’s t-test (vs. YSK-MEND, n=3).

Optimization of Buffer pH and Ionic Strength in Plasmid DNA Encapsulation

To further improve the transgene expression of the YSK-MEND, the ionic make up and buffer pH used in the procedure was optimized. In a previous report, sodium chloride (NaCl) and pH were reported to have a drastic effect on an encapsulation of pDNA.¹⁴⁾ The characteristics and pDNA encapsulation efficiency (EE) were determined for various NaCl concentrations and pH values were used in the preparation (Tables 1, 2). As a result, the concentration and pH of an intermediary lipid/pDNA mixture was at an optimum at pH 6.0 (Fig. 2A). Concerning the NaCl concentration, the amount of pDNA EE was increased in proportion to the amount of NaCl. However, since NaCl crystals were observed in case of concentrations in excess of 200 mM, 100 mM of NaCl was assumed to be the optimum level (Fig. 2B). When pH of the mixture was adjusted to 6.0, an increase in the amount of pDNA had no effect on encapsulation efficiency (Supplementary Figure 1). This was not the case at pH 4.0, however. Under optimal encapsulation conditions, the YSK-MEND showed an approximately 100-fold higher gene expression than that for the original conditions, even though the characteristics of the two carriers were nearly the same (Fig. 2C, Table 2). In addition, expression persisted for at least 1 week (Supplementary Figure 2).

Table 1. Effect of pH on Encapsulation Efficiency and Characteristics of the Carriers

pH	z-Average (nm)	PdI	ζ-Potential (mV)	EE (%)
4.0	109±34	0.39±0.08	−2.7±2.6	66±9
4.5	105±11	0.22±0.05	0.5±1.9	74±13
5.0	103±8	0.25±0.05	−3.8±3.7	80±1
5.5	98±7	0.22±0.06	−1.5±0.7	82±6
6.0	98±3	0.23±0.03	−0.5±0.9	89±3
6.5	95±6	0.18±0.06	1.3±1.9	96±2
7.0	106±12	0.25±0.08	−0.9±1.4	5.0±10

PdI, polydispersity index; EE, encapsulation efficiency.

Table 2. Effect of NaCl Concentration on Encapsulation Efficiency and the Characteristics of the Carriers

NaCl (mM)	z-Average (nm)	PdI	ζ-Potential (mV)	EE (%)
0	103±12	0.20±0.02	−0.7±0.3	79±10
50	106±12	0.22±0.01	−6.8±6.8	85±9
100	108±4	0.18±0.01	−1.4±0.7	87±9
150	103±8	0.24±0.02	−2.0±0.5	86±1
200	95±1	0.24±0.02	−1.9±0.7	92±4
250	108±7	0.20±0.04	−1.0±1.0	94±1
300	92±7	0.22±0.04	−1.7±0.3	89±9
400	98±15	0.18±0.03	−0.7±1.1	94±2
500	109±16	0.27±0.05	0.3±0.5	94±1

PdI, polydispersity index; EE, encapsulation efficiency.

Fig. 2. The Effect of pH and Ionic Strength on pDNA Encapsulation and Transgene Expression in the Liver

A) The encapsulation efficiency of pDNA was evaluated by agarose gel electrophoresis at various pH values (4.0–7.0). B) The encapsulation efficiency of the pDNA was plotted, when NaCl concentration was varied from 0 to 500 mM and pH was fixed at 6.0. C) pDNA delivery efficiency of YSK-MENDs before/after optimization was compared by measuring luciferase expression at a dosage of 10 µg/mouse. Data represents mean and standard deviation. ** p<0.01 Student’s t-test (vs. pH 4.0, NaCl 0 mM, n=3)

Optimization of Lipid Composition of the YSK-MEND

We next examined the effect of the lipid used in the YSK-MEND preparation. Since the lipid/pDNA ratio has a substantial influence on the in vivo transfection efficacy of a pDNA carrier, the optimum nitrogen/phosphate (N/P) ratio was determined. The YSK-MEND showed the highest gene expression when the N/P ratio was 17.5 (Fig. 3). The luciferase expression at an N/P ratio of 17.5 was 10-fold higher than that for the original preparation (N/P ratio of 4.4). The optimum amount of PEG-DMG was next investigated. The characteristics of YSK-MENDs modified with 1–10 mol% PEG-DMG of total lipid were measured. As shown in Figs. 4A and B, the diameter of the YSK-MENDs decreased with increasing PEG-DMG, and the pDNA EE showed a similar decrease. Concerning the delivery of pDNA, a 3 mol% modification was found to result in the highest luciferase activity of these compositions in the liver (Fig. 4C). In the experiment, a YSK-MEND modified with 10 mol% PEG was excluded due to the low EE. The extent of luciferase expression differed from one organ to another (Supplementary Figure 3). Furthermore, as the helper lipid can markedly affect the efficiency of gene delivery,¹⁵⁾ we also examined the effect of additional helper lipid on the process. DPPC or POPE was incorporated into the lipid composition of the YSK-MEND, as these two lipids showed the highest in vitro transfection of the YSK-MEND.⁷⁾ However, no improvement in liver gene expression was observed in either of the cases (Supplementary Figure 4).

Fig. 3. The Effect of N/P Ratio on the Transgene Expression in the Liver

After mice were systemically administered with 5 µg of YSK-MENDs with various N/P ratios (4.4–35), luciferase expression in the liver was determined. Data represents mean and standard deviation. * p<0.05 non-repeated ANOVA followed by Bonferroni test (vs. N/P ratio 4.4, n=3).

Fig. 4. The Effect of PEGylation on the Size and Transgene Expression of YSK-MENDs

A) Particle size distribution of YSK-MENDs with 1–10 mol% PEG-DMG was determined by dynamic light scattering. B) When the PEG modification ratio was varied from 1–10 mol%, the pDNA EE and size were measured. In the graph, the black solid line and the gray dotted line denotes the pDNA EE and number-weighed size, respectively. C) ICR mice were administered with the YSK-MENDs with 1–7 mol% of PEG-DMG at 5 µg/mouse. The luciferase level was determined 24 h after the injection. Data represents mean and standard deviation. * p<0.05 nrANOVA followed by Bonferroni test (vs. PEG 1%, n=3).

Investigation of the Transgene Expression of Optimized YSK-MEND

We then assessed dose-dependent transgene expression for the systemically injected YSK-MEND. The maximum luciferase expression was obtained when the dose administered to the mice was 30 µg/mouse. At higher doses than 30 µg/mouse, about half of the mice died after the injection (data not shown).

Next, to evaluate the expression pattern in the liver, the YSK-MEND encapsulating the pDNA coding fluorescent protein mCherry was administered via the tail vein, and mCherry expression was observed by con-focal laser scanning microscopy (CLSM). The results revealed that the injection of the YSK-MEND resulted in a widespread expression of mCherry in the liver (Fig. 5B), while a HTV injection induced a topical, but very strong fluorescence (Fig. 5C). In addition, the nucleus staining of the HTV injected mice was not strong.

Fig. 5. Transgene Expression of the YSK-MEND

A) Dose–response of YSK-MEND was evaluated. ICR mice were systemically injected with the YSK-MEND with 3 mol% PEG-DMG at doses of 3, 10, 30 µg/mouse, luciferase expression in the liver was measured by means of a luminometer. B), C) To assess a distribution of transgene expression by YSK-MEND injection, pDNA coding mCherry was administered by B) YSK-MEND (10 µg/mouse) or C) HTV injection (0.5 µg/mouse). In the figures, bright circle-shaped dots mean nucleus, and other pale dots indicate mCherry expression.

Toxicological Analysis of the YSK-MEND

Finally, the toxicity of the YSK-MEND was examined. Systemic injection of the YSK-MEND tended to result in the liposomal carriers to accumulate in the liver, as evidenced by measurements of the activity of AST and ALT. A moderate increase in AST and ALT activities were detected only a day after the injection. Nevertheless, AST and ALT activities decreased to normal values 2 d after the treatment (Figs. 6A, B).

Fig. 6. The Hepatological Toxicity of the YSK-MEND

A, B: The activities of the liver escape enzymes, AST and ALT, were measured at 1, 2, 3 and 7 d after the injection. Black solid lines and gray dotted lines denote the YSK-MEND and HTV injection, respectively.

DISCUSSION

In the current study, we report on an efficient method for the encapsulation of pDNA and the effect of preparation method and lipid composition on transgene expression.

pDNA encapsulation was completely different from siRNA encapsulation. Most of the siRNA could be encapsulated without the need for NaCl, but this was not the case for pDNA. In a previous study, pDNA was found to adopt a more compact structure in the presence of NaCl,¹⁶⁾ and therefore pDNA was more efficiently in a compact form in lipid nano particles.¹⁴⁾ Consistent with these reports, it was possible to load the YSK-MEND with up to 85% of pDNA in the presence of 100 mM of NaCl. Regarding pH, 4.5–6.5 was adequate and pH 6.0 was optimum. At a higher pH, YSK05 would not be protonated according to the apparent pK_a of the lipid component (approximately 6.6), which was determined by sodium 6-(p-toluidino)-2-naphthalene sulfonate.⁷⁾ As a large population of the YSK05 molecules are not positively charged at pH 7.0, pDNA could not be able to interact with YSK05 molecules. To examine the effect of pH on EE, we used DOTAP (a quaternary ammonium cation; no pK_a) and DODAP (a tertiary amine; apparent pK_a 5.5). In the case of DODAP, the EE at pH 6.5 was very low (Supplementary Figure 5 and Table 1), whereas it was possible to load the DOTAP-MEND efficiently with pDNA at a high pH. These results are consistent with the above hypothesis. In this context, however, the low EE at an acidic pH is a mystery. Generally, DNA molecules tend to be partitioned in the organic phase in the acid guanidine phenol–chloroform extraction procedure. In a previous study, when pDNA was dissolved in a mixture of hydrophilic PEG₈₀₀₀ and hydrophobic polyacrylate, the pDNA was partitioned in the polyacrylate at a low pH <5.8.¹⁷⁾ This indicates that the hydrophobicity of pDNA is elevated at a low pH. Therefore, it would not be possible to encapsulate the pDNA in the YSK-MEND at a pH<4.0. In addition, the same failure in the case of DOTAP- and DOTAP-based MENDs suggests that the cause of the decrease in EE at low pH can be attributed to pDNA molecules themselves. In addition, encapsulation state of the pDNA plays a key role in the delivery of pDNA to hepatocytes (Figs. 1, 2). This might be because the pDNA is loosely encapsulated at a pH of 4.0 in the absence of NaCl and rapidly leaked from the MENDs in the circulation soon after systemic injection. Moreover, the efficient transgene expression can be attributed to intracellular trafficking. In the case of optimized conditions (pH 6.0, NaCl 100 mM), pDNA was tightly encapsulated even in hepatocytes. Such tightly packaged pDNA could be delivered to the nucleus without being released in the cytosol.

The highest gene expression was realized in the case of a YSK-MEND prepared using 3.0 mol% of PEG-DMG with an N/P ratio of 17.5. In the delivery of nanoparticles to hepatocytes, accessibility through sinusoidal fenestrae from the circulation is obligatory. The fenestrae is a large hole in the liver sinusoidal endothelial cells, the diameter of which ranges from 80–120 nm.¹⁸⁾ Accordingly, a nanocarrier with a small diameter can be delivered to hepatocytes quite efficiently in the case of siRNA delivery.¹⁹⁾ However, higher levels of PEGylation failed to improve transgene expression of YSK-MEND, even when the particle size of the YSK-MENDs was decreased. It is well known that PEG molecules on the surface of a nanocarrier to inhibit the cellular uptake of the carrier and its subsequent endosomal escape.^20,21) Nevertheless, since the PEG–lipid with 14 carbon atoms as an anchor was rapidly removed in the circulation,²²⁾ the effect of PEG on uptake by hepatocytes and endosomal escape would be limited. In terms of pDNA EE, a high level of PEGylation inhibited pDNA loading. As mentioned above, the pDNA EE might drastically affect the delivery of the pDNA. Nevertheless, a 1% PEG modification failed to result in an efficient expression. This is because a YSK-MEND prepared with 1% PEG could not pass through fenestrae due to a large size of the particle. In regard to the N/P ratio, why a N/P ratio of 17.5 was optimal is not known with certainty. More lipid molecules might allow the YSK-MEND to escape from endosomes of hepatocytes and the nuclear import of pDNA could occur via membrane fusion between the nuclear membrane and the YSK-MEND. To the contrary an excessive amount of lipid might disrupt pDNA transcription after its nuclear import because pDNA was transcribed in a naked form. Further study will be required in order to completely elucidate the above results.

Consistent with previous reports,²⁾ HTV injection led to a topical mCherry expression. On the contrary, a homogenous distribution of mCherry was observed in case of a YSK-MEND injection. Although the expression level for YSK-MEND injection is inferior to HTV injection, the homogenous expression would be an advantage in a treatment, for example, for the hepatitis B-type virus.²³⁾ This homogenous expression might be due to an interaction between low density lipoprotein receptor (LDLR) and apolipoprotein E (ApoE) bound to the surface of YSK-MEND. Previously a clearance of neutral liposome was slower in ApoE-deficient mice than that in wild-type mice. This is because ApoE was absorbed to the neutral liposome, and then the absorbed ApoE was endocytosed by LDLR, which was widely distributed protein in hepatocytes. Additionally Akinc et al. recently repoted that siRNA delivery to hepatocytes by lipid nanoparticle (LNP) with neutral charge highly depended on ApoE and LDLR expression.²⁴⁾ Similarly, our YSK-MEND system induced transgene expression in the whole liver through such ApoE-LDLR interaction. However, the hepatic toxicity of the YSK-MEND is not negligible. Actually, the YSK-MEND injection induced the same level of AST and ALT as that for a HTV injection. In contrast, although two picutres were taken in the same condition, the nucleus stain was pale in HTV injection-treated mice. We inferred that the nucleus might be injured by the high physiological pressure accompanied by HTV injection. Since such liver injury was not observed in case of siRNA delivery, this damage can be attributed to a pDNA-derived immune response, such as a stimulator of interferon (IFN) genes (STING), which was known to be a cytosolic DNA sensor.^25,26) To overcome this moderate toxicity associated with the systemic injection of the YSK-MEND, we plan to develop a strategy for relieving a pDNA-derived immune response and the subsequent injury associated with it.

CONCLUSION

A pH-sensitive lipid, YSK05, permitted the efficient encapsulation of plasmid DNA (pDNA) into liposomes. At the preparation step, the pH and ionic strength of the preparation drastically affected, not only the resulting encapsulation efficiency and the characteristics of the particles, but also the transgene expression in the mice liver after a systemic injection. The results demonstrate that pDNA delivery is strongly dependent on the pDNA encapsulation state, but not the nature of the carrier. Both the extent of PEG modification and the nitrogen/phosphate ratio play a key role in the delivery of pDNA to the liver. The YSK05-containing liposome appears to be a safe, efficient carrier for delivering pDNA to hepatocytes in terms of its homogenous nature and high transfection efficiency. In conclusion, YSK05 could be a component of a promising strategy for the treatment of a hepatic disease caused by a genomic disorder.

Acknowledgments

This study was supported in part by Grant-in-Aid for Young Scientists (Start-up) from Japan Society for the Promotion of Science (JSPS) (Grant No. 25893001) and a Grant-in-Aid for Research on Medical Device Development from the Ministry of Health, Labour and Welfare (MHLW) of Japan. We thank Dr. Milton S. Feather for his helpful advice in writing the English manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Park HJ, Yang F, Cho SW. Nonviral delivery of genetic medicine for therapeutic angiogenesis. Adv. Drug Deliv. Rev., 64, 40–52 (2012).
• 2) Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther., 6, 1258–1266 (1999).
• 3) Wells DJ. Opening the floodgates: clinically applicable hydrodynamic delivery of plasmid DNA to skeletal muscle. Mol. Ther., 10, 207–208 (2004).
• 4) Yasuzaki Y, Yamada Y, Kanefuji T, Harashima H. Localization of exogenous DNA to mitochondria in skeletal muscle following hydrodynamic limb vein injection. J. Control. Release, 172, 805–811 (2013).
• 5) Jacobs F, Snoeys J, Feng Y, Van Craeyveld E, Lievens J, Armentano D, Cheng SH, De Geest B. Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer. Gene Ther., 15, 594–603 (2008).
• 6) Suda T, Liu D. Hydrodynamic gene delivery: its principles and applications. Mol. Ther., 15, 2063–2069 (2007).
• 7) Sato Y, Hatakeyama H, Sakurai Y, Hyodo M, Akita H, Harashima H. A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo. J. Control. Release, 163, 267–276 (2012).
• 8) Hayashi Y, Suemitsu E, Kajimoto K, Sato Y, Akhter A, Sakurai Y, Hatakeyama H, Hyodo M, Kaji N, Baba Y, Harashima H. Hepatic monoacylglycerol O-acyltransferase 1 as a promising therapeutic target for steatosis, obesity, and type 2 diabetes. Mol. Ther. Nucleic Acids, 3, e154 (2014).
• 9) Sakurai Y, Hatakeyama H, Akita H, Harashima H. Improvement of doxorubicin efficacy using liposomal anti-polo-like kinase 1 siRNA in human renal cell carcinomas. Mol. Pharm., 11, 2713–2719 (2014).
• 10) Sakurai Y, Hatakeyama H, Sato Y, Hyodo M, Akita H, Ohga N, Hida K, Harashima H. RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal-siRNA system. J. Control. Release, 173, 110–118 (2014).
• 11) Ukawa M, Akita H, Hayashi Y, Ishiba R, Tange K, Arai M, Kubo K, Higuchi Y, Shimizu K, Konishi S, Hashida M, Harashima H. Neutralized nanoparticle composed of SS-cleavable and pH-activated lipid-like material as a long-lasting and liver-specific gene delivery system. Adv. Healthc. Mater., 3, 1222–1229 (2014).
• 12) Khalil IA, Hayashi Y, Mizuno R, Harashima H. Octaarginine- and pH sensitive fusogenic peptide-modified nanoparticles for liver gene delivery. J. Control. Release, 156, 374–380 (2011).
• 13) Sakurai Y, Hatakeyama H, Sato Y, Hyodo M, Akita H, Harashima H. Gene silencing via RNAi and siRNA quantification in tumor tissue using MEND, a liposomal siRNA delivery system. Mol. Ther., 21, 1195–1203 (2013).
• 14) Jeffs LB, Palmer LR, Ambegia EG, Giesbrecht C, Ewanick S, MacLachlan I. A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA. Pharm. Res., 22, 362–372 (2005).
• 15) Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J. Control. Release, 116, 255–264 (2006).
• 16) Schlick T, Li B, Olson WK. The influence of salt on the structure and energetics of supercoiled DNA. Biophys. J., 67, 2146–2166 (1994).
• 17) Johansson HO, Matos T, Luz JS, Feitosa E, Oliveira CC, Pessoa A Jr, Bulow L, Tjerneld F. Plasmid DNA partitioning and separation using poly(ethylene glycol)/poly(acrylate)/salt aqueous two-phase systems. J. Chromatogr. A, 1233, 30–35 (2012).
• 18) Jacobs F, Wisse E, De Geest B. The role of liver sinusoidal cells in hepatocyte-directed gene transfer. Am. J. Pathol., 176, 14–21 (2010).
• 19) Akinc A, Goldberg M, Qin J, Dorkin JR, Gamba-Vitalo C, Maier M, Jayaprakash KN, Jayaraman M, Rajeev KG, Manoharan M, Koteliansky V, Rohl I, Leshchiner ES, Langer R, Anderson DG. Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol. Ther., 17, 872–879 (2009).
• 20) Harvie P, Wong FM, Bally MB. Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles. J. Pharm. Sci., 89, 652–663 (2000).
• 21) Hatakeyama H, Akita H, Harashima H. A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma. Adv. Drug Deliv. Rev., 63, 152–160 (2011).
• 22) Mui BL, Tam YK, Jayaraman M, Ansell SM, Du X, Tam YY, Lin PJ, Chen S, Narayanannair JK, Rajeev KG, Manoharan M, Akinc A, Maier MA, Cullis P, Madden TD, Hope MJ. Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Mol. Ther. Nucleic Acids, 2, e139 (2013).
• 23) Sato S, Li K, Kameyama T, Hayashi T, Ishida Y, Murakami S, Watanabe T, Iijima S, Sakurai Y, Watashi K, Tsutsumi S, Sato Y, Akita H, Wakita T, Rice CM, Harashima H, Kohara M, Tanaka Y, Takaoka A. The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus. Immunity, 42, 123–132 (2015).
• 24) Akinc A, Querbes W, De S, Qin J, Frank-Kamenetsky M, Jayaprakash KN, Jayaraman M, Rajeev KG, Cantley WL, Dorkin JR, Butler JS, Qin L, Racie T, Sprague A, Fava E, Zeigerer A, Hope MJ, Zerial M, Sah DW, Fitzgerald K, Tracy MA, Manoharan M, Koteliansky V, Fougerolles Ad, Maier MA. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms. Mol. Ther., 18, 1357–1364 (2014).
• 25) Cai X, Chiu YH, Chen ZJ. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol. Cell, 54, 289–296 (2014).
• 26) Civril F, Deimling T, de Oliveira Mann CC, Ablasser A, Moldt M, Witte G, Hornung V, Hopfner KP. Structural mechanism of cytosolic DNA sensing by cGAS. Nature, 498, 332–337 (2013).