Atsushi Harada - Cochemist

Gene expression of ternary complexes through the compaction of nanofiber-polyplexes by mixing with lipofectamine

Co-reporter:Ryuta Aono, Kenta Nomura, Eiji Yuba, Atsushi Harada and Kenji Kono

Biomaterials Science 2015 vol. 3(Issue 5) pp:764-770

Publication Date(Web):02 Apr 2015

DOI:10.1039/C5BM00081E

For the development of an effective nonviral gene vector, ternary complexes were prepared through the compaction of nanofiber-polyplexes. These were formed using pDNA and a head-tail type polycation bearing a multi-arm poly(ethylene glycol) head and a poly(L-lysine) tail, and this strategy was based on the crowding effect of poly(ethylene glycol) in the polyplex. Mixing was carried out using a cationic lipid (lipofectamine), which is a commercially available transfection reagent. Through ternary complex formation, the elongated morphology of nanofiber-polyplexes was found to compact into a spherical shape with an average diameter of ca. 100 nm. Accompanying ternary complex formation, the compaction of the nanofiber-polyplexes can improve cellular uptake and helps the ternary complex to retain its smooth transcription/translation process, which is characteristic of nanofiber-polyplexes. As a result, ternary complexes prepared at an optimal mixing ratio exhibit a high transfection efficiency compared with lipofectamine lipoplexes.

Effective Condensation of Multivalent Anions into Polyion Complex Micelles Prepared from TiO2 Nanoparticles and Polyallylamine Bearing Poly(ethylene glycol) Grafts

Co-reporter:Satoshi Yamamoto, Eiji Yuba, Atsushi Harada, and Kenji Kono

Langmuir 2015 Volume 31(Issue 31) pp:8583-8588

Publication Date(Web):July 17, 2015

DOI:10.1021/acs.langmuir.5b01883

Physicochemical properties were evaluated for polyion complex micelles (PIC), which were prepared from TiO2 nanoparticles and polyallylamine bearing poly(ethylene glycol) grafts (PAA-g-PEG). The zeta potentials of PIC micelles prepared using PAA-g-PEG with different molecular weights of PEG grafts were measured in different aqueous media (i.e., water, phosphate buffer, and Tris/HCl buffer). The PIC micelles in phosphate buffer and Tris/HCl buffer exhibited quite different zeta potentials despite the same salt concentration (10 mM) of the buffer solutions. More specifically, the zeta potential of the PIC micelles in phosphate buffer was effectively neutralized owing to counteranion condensation effects. The onset of counterion condensation into the PIC micelles was dependent on the valence of the anionic molecules and the ability of the PIC micelles to entrap multivalent anionic molecules. Furthermore, as confirmed by laser confocal microscopy observation, multivalent anionic molecules could be delivered to cultured cells through entrapment in the PIC micelles based on multivalent anion condensation effects.

Nanofiber Polyplex Formation Based on the Morphology Elongation by the Intrapolyplex PEG Crowding Effect

Co-reporter:Ryuta Aono, Eiji Yuba, Atsushi Harada, and Kenji Kono

ACS Macro Letters 2014 Volume 3(Issue 4) pp:333

Publication Date(Web):March 24, 2014

DOI:10.1021/mz500072k

We prepared a multiarm poly(ethylene glycol)-poly(l-lysine) block copolymer (maPEG-PLL) with a size-controllable maPEG head and a cationic PLL tail for the evaluation of the effect of maPEG crowding to the polyplex formation with plasmid DNA. maPEG-PLLs of various compositions were synthesized and the formation of a polyplex was confirmed by gel retardation assay. The maPEG-PLL exhibited noncooperative polyplex formation behavior, suggesting the effective hydration of the polyplex. Also, an increase in the size of the maPEG head induces the elongation of polyplex morphology from spherical aggregates to nanorods and nanofibers because of the intrapolyplex PEG crowding effect. Furthermore, an increase in the size of the maPEG head also improves the effective inhibition of the decrease in cell-free gene expression, indicating the importance of the control of pDNA packaging in the polyplex.

Titanium dioxide nanoparticle-entrapped polyion complex micelles generate singlet oxygen in the cells by ultrasound irradiation for sonodynamic therapy

Co-reporter:Atsushi Harada, Masafumi Ono, Eiji Yuba and Kenji Kono

Biomaterials Science 2013 vol. 1(Issue 1) pp:65-73

Publication Date(Web):26 Sep 2012

DOI:10.1039/C2BM00066K

A new modality of using ultrasound irradiation instead of photoactivation, as in photodynamic therapy (PDT), sonodynamic therapy, has emerged as a promising treatment for various types of cancer. Titanium dioxide (TiO2) has the ability to generate reactive oxygen species (ROS) by not only photo- but also ultrasound-irradiation. Here, the formation of core–shell type polyion complex micelles from TiO2 nanoparticles with polyallylamine bearing poly(ethylene glycol) grafts effectively improves the dispersion stability of the TiO2 nanoparticles under physiological conditions for therapeutic application. The TiO2 nanoparticles in the micelles can generate ROS including singlet oxygen (1O2) by sonication. Furthermore, the micelles are taken up into HeLa cells and the TiO2 nanoparticles generate 1O2, which is widely believed to be the main cytotoxic agent in PDT, even in the cells treated by sonication. This is the first result representing 1O2 generation of TiO2 nanoparticles in HeLa cells by sonication. Further, the micelles can selectively exhibit a cell-killing effect at only the ultrasound-irradiated area.

Hollow nanocapsules prepared through stabilization of polymer vesicles formed from head–tail type polycations by introducing cross-linkages

Co-reporter:Atsushi Harada, Shin-ichi Ichimura, Eiji Yuba and Kenji Kono

Soft Matter 2011 vol. 7(Issue 10) pp:4629-4635

Publication Date(Web):13 Apr 2011

DOI:10.1039/C1SM05075C

Hollow nanocapsules were prepared through introduction of cross-linkages into polymer vesicles formed from head–tail type polycations composed of polyamidoamine dendron heads and poly(L-lysine) (PLL) tails. The PLL tails were cross-linked by reaction of lysine residues with oligo(ethylene glycol) bearing epoxy groups at both ends. Successful fixing of hollow structures by crosslinking was confirmed by dynamic light scattering (DLS) measurements and transmission electron microscopy observation. The pH-dependent properties of the nanocapsules were evaluated by DLS, ζ-potential, fluorescence and circular dichroism measurements. The increase in the size (i.e. swelling) of the nanocapsules resulted from collapse of the helical conformation of PLL tails in response to decreased pH or addition of urea, indicating that the conformation of PLL tails was a crucial factor in determining the size of the nanocapsules.

Effect of pH and generation of dendron on single-step synthesis of gold nanoparticles using PEGylated polyamidoamine dendron in aqueous medium

Co-reporter:Atsushi Harada;Akihiko Yuzawa;Takeshi Kato;Chie Kojima ;Kenji Kono

Journal of Polymer Science Part A: Polymer Chemistry 2010 Volume 48( Issue 6) pp:1391-1398

Publication Date(Web):

DOI:10.1002/pola.23901

Abstract

Three types of PEGylated polyamidoamine (PAMAM) dendrons were synthesized through PEGylation of primary amines at the periphery of second, third, and fourth generation dendrons. Au(III) precursors and the synthesized PEGylated PAMAM dendrons were mixed at various pHs to evaluate the effect of pH on gold nanoparticle (Au NP) synthesis by monitoring the change in surface plasmon resonance. The Au NP synthesis reaction was controlled by pH through the balance between protonated and deprotonated tertiary amines and the reactivity of Au(III) precursors. By using PEGylated PAMAM dendrons with higher generation, the obtained Au NPs had narrow size distribution with small average size because of the limitation of intermolecular space among PEGylated PAMAM dendrons for the growth to Au NP. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1391–1398, 2010

Cationic Polymers with Inhibition Ability of DNA Condensation Elevate Gene Expression

Co-reporter:Dr. Atsushi Harada;Yuka Kimura ; Kenji Kono

ChemBioChem 2010 Volume 11( Issue 14) pp:1985-1988

Publication Date(Web):

DOI:10.1002/cbic.201000394

Effective Tolerance to Serum Proteins of Head−Tail Type Polycation Vectors by PEGylation at the Periphery of the Head Block

Co-reporter:Atsushi Harada, Yuka Kimura, Chie Kojima and Kenji Kono

Biomacromolecules 2010 Volume 11(Issue 4) pp:

Publication Date(Web):March 4, 2010

DOI:10.1021/bm1000108

To improve the stability of a head−tail type polycation (PAMAM dendron-PLL), which is composed of a polyamidoamine (PAMAM) dendron head and poly(l-lysine) (PLL) tail blocks, as a gene vector against serum proteins, the PEGylation of the PAMAM dendron head was examined using physicochemical and transfection experiments. The PEGylation of the PAMAM dendron-PLL polyplexes neutralized the zeta-potential, indicating that the PEG brushes surround the surface of the polyplexes. The PEGylated PAMAM dendron-PLL polyplexes had higher tolerance against incubation with serum proteins when compared with non-PEGylated PAMAM dendron-PLL polyplexes. In addition, the transfection efficiency of the PEGylated PAMAM dendron-PLL polyplexes increased with increasing incubation periods, leading to an increase in the amount of pDNA uptaked by HeLa cells. Furthermore, effective stabilization of the PAMAM dendron-PLL polyplexes through PEGylation of the PAMAM dendron block was confirmed by the transfection experiments after preincubation with serum proteins and the PEGylated PAMAM dendron-PLL polyplexes showed a comparable level of transfection efficiency with polyethyleneimine polyplexes.

Effect of Head Size in Head-Tail-Type Polycations on their in vitro Performances as Nonviral Gene Vectors

Co-reporter:Atsushi Harada;Masanori Kawamura;Yuka Kimura;Toshinari Takahashi;Chie Kojima ;Kenji Kono

Macromolecular Bioscience 2009 Volume 9( Issue 6) pp:605-612

Publication Date(Web):

DOI:10.1002/mabi.200800314

Spontaneous formation of narrowly-distributed self-assembly from polyamidoamine dendron-poly(L-lysine) block copolymers through helix-coil transition of poly(L-lysine) block

Co-reporter:Atsushi Harada;Kenichi Nakanishi;Shinichi Ichimura;Chie Kojima ;Kenji Kono

Journal of Polymer Science Part A: Polymer Chemistry 2009 Volume 47( Issue 4) pp:

Publication Date(Web):

DOI:10.1002/pola.23231

Abstract

The self-assembly of head-tail type block copolymers composed of polyamidoamine dendron head block and poly(L-lysine) (PLL) tail block was studied using a light scattering technique and transmission electron microscopy. A PLL tail block in a head-tail type block copolymer exhibits a coil-to-helix transition as a result of the change in solvent quality from water to methanol. When the PLL tail block takes a helical conformation in high methanol content, the resulting head-tail type block copolymer has a defined three-dimensional structure like that of a protein molecule. Self-assemblies of such block copolymers having a totally fixed molecular shape spontaneously form polymersome-like self-assemblies with an extremely narrow size distribution through converging to a thermodynamically stable assembling state. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1217–1223, 2009

Polyion complex micelles formed from glucose oxidase and comb-type polyelectrolyte with poly(ethylene glycol) grafts

Co-reporter:Akifumi Kawamura;Chie Kojima;Michihiro Iijima;Kenji Kono

Journal of Polymer Science Part A: Polymer Chemistry 2008 Volume 46( Issue 11) pp:3842-3852

Publication Date(Web):

DOI:10.1002/pola.22739

Abstract

The comb-type polyelectrolyte, poly(ethylene glycol)-graft-poly(allyl amine) (PEG-g-PAA), was synthesized to prepare polyion complex (PIC) micelles with Aspergillus Niger Glucose oxidase (GOD). Even after mixing GOD and PEG-g-PAAs with various PEG contents, the resulting mixtures remained transparent but the mixture of GOD and PAA homopolymer immediately precipitated. In the mixtures prepared with a stoichiometric mixing ratio, the formation of PIC micelles with a core-shell structure was suggested from dynamic and static light scattering measurements. Glucose, the substrate for GOD, could easily diffuse into the PIC micelles, and the GOD molecules were active even in the core of the PIC micelles. GOD didn't lose its enzymatic activity through entrapment into the PIC micelles. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3842–3852, 2008

Effect of Polycarboxylate Blocks on the Amidase Activity of Trypsin through Complexation with PEG/Polycarboxylate Block Ionomers

Co-reporter:Chie Kojima;Yuriko Yoshioka;Akifumi Kawamura;Kenji Kono

Macromolecular Bioscience 2007 Volume 7(Issue 3) pp:339-343

Publication Date(Web):16 MAR 2007

DOI:10.1002/mabi.200600199

The amidase reaction of trypsin, which is a member of the serine proteinase family, is accelerated by its complexation with block ionomers containing a polycarboxylate block, such as PEG-PAA, PEG-PGA, or PEG-PMA. PEG-PAA and PEG-PGA had similar effects, causing an increase in the k_cat value and a shift in the pH profile to a lower pH region. On the other hand, PEG-PMA showed not only an increase in the k_cat value, but also a decrease in the activation energy; however, there was no shift in the pH dependence of the initial reaction rate. Such differences might be induced by the difference in pK_a values of the polycarboxylate block in block ionomers.

Preparation of temperature-responsive polymer gels physically immobilizing core-shell type bioconjugates

Co-reporter:Atsushi Harada;Kunio Johnin;Kenji Kono;Akifumi Kawamura

Journal of Polymer Science Part A: Polymer Chemistry 2007 Volume 45(Issue 24) pp:5942-5948

Publication Date(Web):2 NOV 2007

DOI:10.1002/pola.22347

Poly(N-isopropylacrylamide) (PNIPAAm) gels are temperature-responsive polymer gels; and were prepared by redox polymerization of N-isopropylacrylamide in the presence of N,N′-methylenebisacrylamide as a crosslinking reagent and core-shell type bioconjugates, which were core-crosslinked polyion complex micelles formed from the mixture of bovine pancreas trypsin and poly(ethylene glycol)-block-poly(α,β-aspartic acid). The phase transition temperature of PNIPAAm gels was no change with physically immobilization of bioconjugates. Also, the enzymatic activity of bioconjugates was essentially maintained even in PNIPAAm gels, although enzymatic reaction rate was apparently controlled by temperature, i.e., by the degree of swelling of PNIPAAm gels. Further, the control of enzymatic reaction synchronizing the phase transition of PNIPAAm gels immobilized bioconjugates. PNIPAAm gels could immobilize core-shell type bioconjugates, and were successfully prepared without interfering with the properties of temperature-responsive polymer gels and the bionanoreactor. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5942–5948, 2007

Gene expression of ternary complexes through the compaction of nanofiber-polyplexes by mixing with lipofectamine

Co-reporter:Ryuta Aono, Kenta Nomura, Eiji Yuba, Atsushi Harada and Kenji Kono

Biomaterials Science (2013-Present) 2015 - vol. 3(Issue 5) pp:NaN770-770

Publication Date(Web):2015/04/02

DOI:10.1039/C5BM00081E

For the development of an effective nonviral gene vector, ternary complexes were prepared through the compaction of nanofiber-polyplexes. These were formed using pDNA and a head-tail type polycation bearing a multi-arm poly(ethylene glycol) head and a poly(L-lysine) tail, and this strategy was based on the crowding effect of poly(ethylene glycol) in the polyplex. Mixing was carried out using a cationic lipid (lipofectamine), which is a commercially available transfection reagent. Through ternary complex formation, the elongated morphology of nanofiber-polyplexes was found to compact into a spherical shape with an average diameter of ca. 100 nm. Accompanying ternary complex formation, the compaction of the nanofiber-polyplexes can improve cellular uptake and helps the ternary complex to retain its smooth transcription/translation process, which is characteristic of nanofiber-polyplexes. As a result, ternary complexes prepared at an optimal mixing ratio exhibit a high transfection efficiency compared with lipofectamine lipoplexes.

Titanium dioxide nanoparticle-entrapped polyion complex micelles generate singlet oxygen in the cells by ultrasound irradiation for sonodynamic therapy

Co-reporter:Atsushi Harada, Masafumi Ono, Eiji Yuba and Kenji Kono

Biomaterials Science (2013-Present) 2013 - vol. 1(Issue 1) pp:NaN73-73

Publication Date(Web):2012/09/26

DOI:10.1039/C2BM00066K

A new modality of using ultrasound irradiation instead of photoactivation, as in photodynamic therapy (PDT), sonodynamic therapy, has emerged as a promising treatment for various types of cancer. Titanium dioxide (TiO2) has the ability to generate reactive oxygen species (ROS) by not only photo- but also ultrasound-irradiation. Here, the formation of core–shell type polyion complex micelles from TiO2 nanoparticles with polyallylamine bearing poly(ethylene glycol) grafts effectively improves the dispersion stability of the TiO2 nanoparticles under physiological conditions for therapeutic application. The TiO2 nanoparticles in the micelles can generate ROS including singlet oxygen (1O2) by sonication. Furthermore, the micelles are taken up into HeLa cells and the TiO2 nanoparticles generate 1O2, which is widely believed to be the main cytotoxic agent in PDT, even in the cells treated by sonication. This is the first result representing 1O2 generation of TiO2 nanoparticles in HeLa cells by sonication. Further, the micelles can selectively exhibit a cell-killing effect at only the ultrasound-irradiated area.