Tomoya Masuda

Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

Evaluation of nuclear transfer and transcription of plasmid DNA condensed with protamine by microinjection: The use of a nuclear transfer score

In the present study, the nuclear delivery of a green fluorescence protein (GFP)‐encoding pDNA condensed by protamine was investigated in terms of trans‐gene expression after cytoplasmic (E(cyt)) and nuclear (E(nuc)) microinjection. To compare the nuclear transfer process, a novel parameter; the nuclear transfer (NT) score was introduced. The E(cyt) value for protamine/pDNA particles increased in a charge ratio‐dependent manner. The calculated NT score showed that this increase results from an enhancement in nuclear transfer efficiency, which was also quantitatively confirmed by a recently developed confocal image‐assisted three‐dimensionally integrated quantification (CIDIQ) method. Moreover, E(nuc) for protamine/pDNA particles was significantly higher than that for poly‐l‐lysine/pDNA particles, suggesting that pDNA, when condensed with protamine, is more accessible to intra‐nuclear transcription. Collectively, protamine is an excellent DNA condenser, with bi‐functional advantages: improvement in nuclear delivery and efficient intra‐nuclear transcription.

Enhanced gene expression by a novel stearylated INF7 peptide derivative through fusion independent endosomal escape

An octaarginine-modified multifunctional envelope-type nano device (R8-MEND) was previously reported to be an efficient nonviral vector for the delivery of plasmid DNA, in vitro and after topical administration. We report herein on a novel stearylated derivative of the INF7 peptide, a derivative of the N-terminal domain of the HA2 protein of the influenza virus envelope, which enhances the endosomal escape of R8-MEND through a mechanism independent of fusion between the MEND coat and the endosomal membrane. The use of the novel peptide derivative would permit the gene expression of the R8-MEND to be improved, both in vitro and in vivo. R8-MEND modified with stearylated INF7 resulted in gene expression levels that were 77-fold higher than unmodified and 20-fold higher than the free INF7 peptide-modified R8-MEND with no cellular toxicity. Spectral imaging in live cells confirmed that the stearylated INF7 modification did not mediate fusion between liposomes and the endosomal membrane. The inclusion of DOPE to the R8-MEND coat was synergistic with the peptide in improving gene transfection. The intravenous injection of an R8-MEND modified with stearylated INF7 to ICR mice resulted in luciferase expression levels 240-fold higher in liver and 115-fold higher in spleen than that of the R8-MEND.

Evaluation of the nuclear delivery and intra-nuclear transcription of plasmid dna condensed with µ (mu) and nls-µ by cytoplasmic and nuclear microinjection: a comparative study with poly-l-lysine

The efficient nuclear delivery of plasmid DNA (pDNA) is essential for the development of a promising non‐viral gene vector. In an attempt to achieve nuclear delivery, NLS‐mu, a novel pDNA condenser, was prepared. This consists of mu, a highly potent polypeptide for condensing the pDNA, and a SV40 T antigen‐derived nuclear localization signal (NLSSV40).

2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes.

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the zeta-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Envelope-type lipid nanoparticles incorporating a short PEG-lipid conjugate for improved control of intracellular trafficking and transgene transcription.

Lipid envelope-type nanoparticles are promising carriers for gene delivery. The modification of liposomes with polyethyleneglycol (PEG) can often be useful in liposomal formation and pharmacokinetics. However, there is a dilemma concerning the use of PEG because of its poor intracellular trafficking properties. To overcome this problem, in the present study, we report on a strategy for improving the intracellular trafficking of PEG-modified lipid particles by incorporating a short PEG lipid. The findings presented here show that the incorporation of tetra(ethylene)glycol (TEG)-conjugated cholesterol into a liposome composition is useful in controlling the number of lipid envelopes, resulting in an improvement in particle uniformity with a reduced particle size. The TEG-modified lipid particles were found to enhance transfection activity by more than 100-fold. This increase is attributed to an enhancement of cellular uptake, and nuclear transcription by improving intracellular decoating. Moreover, the use of a various short PEG lipids in lipid particle formation showed a clear threshold polymerization degree (less or equal 25: PEG1100), for achieving stimulated transfection activity. Collectively, the use of short PEG lipid promises to be useful in developing an efficient non-viral gene vector.

Particle Tracking of Intracellular Trafficking of Octaarginine-modified Liposomes: A Comparative Study With Adenovirus

It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)-tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips.

Oligosaccharide‐Mediated Nuclear Transport of Nanoparticles

The transport of nanoparticles into the cellular nucleus is a potentially important technique because it can open the way to a wide range of applications, including the sequence-specific detection of genomic DNA, efficient DNA transfection, and the specific entry of drugs into the nucleus. It has been reported that the nuclear import of proteins larger than 40 kD does not occur by passive diffusion. Similarly, the nuclear import of macromolecules or particles is strictly regulated. Therefore, the nuclear import of nanoparticles that contain gold nanoparticles and quantum dots has been achieved by coating the surface with classical nuclear localization signals (NLS), that is, short, highly positively charged peptides. However, the problem remains that positively charged particles can interact with serum protein, resulting in rapid clearance from the plasma compartment. Because the cationic NLS interacts with negatively charged DNA, NLS peptides do not work as efficient signals for transport of DNA into the nucleus; this implies that the use of peptide-based cationic NLS might be limited when using DNA-displaying nanoparticles. Monsigny et al. have shown that sugars can also work as nuclear localization signals. The neoglycoproteins, BSA (bovine serum albumin)–glucose, BSA–mannose and BSA–fucose are rapidly transported into the nucleus of HeLa cells, whereas BSA without chemical modifications is not. Because carbohydrates normally show high biocompatibility and water solubility, they are suitable for use in the modification of synthetic carriers and nanoparticles. Previously, however, only the application of these carbohydrate signals to the nuclear import of proteins was examined, and there are no reports on the effectiveness of carbohydrate signals for the nuclear import of artificial materials, such as nanoparticles and polymers. Previous reports that used BSA have focused only on monosaccharides as a signal. In this paper, we expand the varieties of carbohydrates tested from monosaccharides to oligosaccharides in the search for an efficient signal that is applicable to the nuclear import of nanoparticles. Herein, we present our unique finding that nanoparticles (quantum dots) that display oligo a-glucopyranoside on their surface are readily transported into the nucleus of digitonin-permeabilized HeLa cells. Semiconductor QDs have a diameter of several nanometers and their specific transport inside the cell can be readily achieved through the display of multiple ligands on their surface. As far as we know, this is the first report to describe the import of nanoparticles into the nucleus without the use of cationic NLS. Because BSA that has been substituted with a-glucopyranoside has been reported to be efficiently transported into the cell nucleus, we synthesized neoglycolipids that contain various carbohydrates comprised of different numbers of glucose units (Scheme 1). In addition to a-monoglucopyranoside–lipid 3, we synthesized maltose(Glca1-4Glc)–lipid 4, maltotriose(Glca1-4Glca1-4Glc)–lipid 5, and panose(Glca1-6Glca1-4Glc)–lipid 6. The cellotriose(Glcb1-4Glcb1-4Glc)–lipid 7 is a trisaccharide that is composed of only a b-linked glucose, thus lipid 7 was used as a control to a-linked glucose. Neoglycolipids were synthesized based on a previously described method 11] by starting from fully acetylated carbohydrates, hexa(ethyleneglycol) and 11-bromoundecene. CdTe nanocrystals that had been stabilized with mercaptopropionic acid (MPA) were prepared in water as described by Yang et al. Sugar-displaying CdTe QDs were synthesized by surface exchange from MPA to the neoglycolipid in water (Figure 1A), and then purified by using spin filtration. Ligand exchange occurring on the surface of the QDs was confirmed by MALDI-ToF mass spectrometry. Ligands immobilized on inorganic nanoparticles, such as QDs, were detached from the surface during the laser deposition process, and the mass corresponding to the molecular weight of neoglycolipids was clearly detected (Figure 1B). Furthermore, the display of sugars on the QDs was visually confirmed by trapping on a lectin-immobilized column. For example, maltotriose 5–QDs were specifically trapped on a ConA (concanavalin A; a-mannose and a-glucose specific) agarose column, whereas they were not trapped on WGA (wheat germ agglutinin; GlcNAc specific) or LCA (lens culinaris agglutinin; branched-fucose specific) agarose columns (data not shown). We explored the interactions between sugar-displaying CdTe QDs and digitonin-permeabilized HeLa cells. Digitonin treatment causes partial damage to the plasma membrane and increases the permeability of cells. Digitonin permeabilization has been used often in the study of biochemical processes that are related to the import and export of nuclear proteins. Although live cells become semi-intact upon digitonin treatment due to the leakage of cytoplasmic proteins through the plasma membrane, the nuclear membrane is left intact. Import buffer (pH 7.3, 20 mm HEPES, 110 mm KOAc, 5 mm NaOAc, 2 mm MgACHTUNGTRENNUNG(OAc)2, 0.5 mm EGTA) that contained the sugar-displaying CdTe QDs (0.6 mgmL , [a] Dr. K. Niikura, Dr. Y. Matsuo, Dr. K. Ijiro Research Institute for Electronic Science, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) Fax: (+81)11-706-9361 E-mail : kniikura@poly.es.hokudai.ac.jp ijiro@poly.es.hokudai.ac.jp [b] S. Sekiguchi, T. Nishio Department of Chemistry, Hokkaido University N21W10, Kita-ku, Sapporo 001-0021 (Japan) [c] T. Masuda, Dr. H. Akita, Dr. H. Harashima Faculty of Pharmaceutical Science, Hokkaido University N12 W6, Kita-Ku, Sapporo 060-0812 (Japan) [d] Dr. K. Kogure Department of Biophysical Chemistry, Kyoto Pharmaceutical University Misasagi-Nakauchicho 5, Yamashinaku, Kyoto 607-84142 (Japan)

Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose.

The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.

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.