Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly­(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly­(2-<i>n</i>-propyl-2-oxazoline) (PnPrOx) and cationic poly­(l-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic–hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly­(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications

Intravenous injection of psFlt-1-PM decreased the area of CNV.

<p>Quantification of the CNV lesion demonstrated that the neovascularized area in the mice that received psFlt-1-PM was significantly reduced by 60% than that of control mice (n = 7, p<0.01), whereas the administration of pYFP-PM had no significant effects on the neovascularized area. The lower panels show representative micrographs. Arrows indicate the CNV lesion. CNV: choroidal neovascularization, psFlt-1-PM: the PIC micelle encapsulating psFlt-1 (fms-like tyrosine kinase-1), pYFP-PM: the PIC micelles encapsulating pYFP (yellow fluorescent protein).</p

Targeted gene expression in the CNV area after intravenous injection of pYFP-PM.

<p>a: Choroidal flatmount and histological analysis demonstrated YFP fluorescence in the CNV area after intravenous injection of pYFP-PM. b: Western blotting analysis demonstrated that YFP protein was detected in the eyes with CNV, after intravenous injection of pYFP-PM. YFP expression was detected neither after generation of CNV alone nor after intravenous injection of pYFP-PM without CNV. c: The results of immunohistochemistry demonstrated that the expression of F4/80 was partially overlapping the expression of YFP, indicating that YFP protein expressed in F4/80-positive macrophage. CNV: choroidal neovascularization, pYFP-PM: the PIC micelles encapsulating pYFP (yellow fluorescent protein), PC: photocoagulation, bar:100 um.</p

Laboratory data.

<p>Laboratory data.</p

Intracellular Delivery of Charge-Converted Monoclonal Antibodies by Combinatorial Design of Block/Homo Polyion Complex Micelles

Direct intracellular delivery of antibodies has gained much attention, although only a few agents have been developed, and none of them has reached clinical stages. The main obstacles here are the insufficient characteristics of delivery systems including stability and appropriate ability for intracellular antibody release. We tailored the structure of polyion complex (PIC) micelles by loading transiently charge-converted antibody derivatives for achieving enhanced stability, delivery to cytosol, and precise antigen recognition inside cells. Citraconic anhydride was used for the charge conversion of the antibody; the optimized degree of modification was identified to balance the stability of PIC micelles in the extracellular compartment and prompt pH-triggered disintegration after their translocation into the acidic endosomal compartment of target cells. The use of a mixture of homo- and block-catiomers in an appropriate ratio to construct PIC micelles substantially enhanced the endosomal escaping efficacy of the loaded antibody, leading to improved recognition of intracellular antigens

Polyplex Micelles with Phenylboronate/Gluconamide Cross-Linking in the Core Exerting Promoted Gene Transfection through Spatiotemporal Responsivity to Intracellular pH and ATP Concentration

Polyplexes as gene delivery carriers require integrated functionalities to modulate intracellular trafficking for efficient gene transfection. Herein, we developed plasmid DNA (pDNA)-loaded polyplex micelles (PMs) from poly­(ethylene glycol)-based block catiomers derivatized with 4-carboxy-3-fluoro­phenyl­boronic acid (FPBA) and d-glucon­amide to form pH- and ATP-responsive cross-linking in the core. These PMs exhibited robustness in the extracellular milieu and smooth endosomal escape after cellular uptake, and they facilitated pDNA decondensation triggered by increased ATP concentration inside of the cell. Laser confocal microscopic observation revealed that FPBA installation enhanced the endosomal escapability of the PMs; presumably, this effect resulted from the facilitated endo-/lysosomal membrane disruption triggered by the released block catiomers with hydrophobic FPBA moieties in the side chain from the PM at lower pH condition of endo-/lysosomes. Furthermore, the profile of intracellular pDNA decondensation from the PMs was monitored using Förster resonance energy transfer measurement by flow cytometry; these observations confirmed that PMs optimized for ATP-responsivity exerted effective intracellular decondensation of loaded pDNA to attain promoted gene transfection

<em>In Vivo</em> Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle

<div><p>Messenger RNA (mRNA) introduction is a promising approach to produce therapeutic proteins and peptides without any risk of insertion mutagenesis into the host genome. However, it is difficult to introduce mRNA <em>in vivo</em> mainly because of the instability of mRNA under physiological conditions and its strong immunogenicity through the recognition by Toll-like receptors (TLRs). We used a novel carrier based on self-assembly of a polyethylene glycol (PEG)-polyamino acid block copolymer, polyplex nanomicelle, to administer mRNA into the central nervous system (CNS). The nanomicelle with 50 nm in diameter has a core-shell structure with mRNA-containing inner core surrounded by PEG layer, providing the high stability and stealth property to the nanomicelle. The functional polyamino acids possessing the capacity of pH-responsive membrane destabilization allows smooth endosomal escape of the nanomicelle into the cytoplasm. After introduction into CNS, the nanomicelle successfully provided the sustained protein expression in the cerebrospinal fluid for almost a week. Immune responses after mRNA administration into CNS were effectively suppressed by the use of the nanomicelle compared with naked mRNA introduction. <em>In vitro</em> analyses using specific TLR-expressing HEK293 cells confirmed that the nanomicelle inclusion prevented mRNA from the recognition by TLRs. Thus, the polyplex nanomicelle is a promising system that simultaneously resolved the two major problems of <em>in vivo</em> mRNA introduction, the instability and immunogenicity, opening the door to various new therapeutic strategies using mRNA.</p> </div

Comparison of mRNA, plasmid DNA (pDNA) and protein.

<p>mRNA or pDNA that expressed GLuc was incorporated in the polyplex nanomicelle and injected into the subarachnoid space of rats by intrathecal injection. The proteinous form of GLuc was also used for intrathecal injection. GLuc expression was evaluated from cerebrospinal fluid (CSF) collected at the indicated time points. The data are presented as the mean ± standard error of the mean (s.e.m.) (N = 4). RLU, relative luminescence units.</p

Synthetic Polyamines to Regulate mRNA Translation through the Preservative Binding of Eukaryotic Initiation Factor 4E to the Cap Structure

Polyion complexes (PICs) of mRNA with synthetic polyamines are receiving increasing attention as mRNA delivery vehicles, and the search for polyamine structure maximizing the translational efficiency of complexed mRNA becomes a critical research topic. Herein, we discovered that fine-tuning of the protonation status of synthetic polyamines can regulate mRNA translation through the preservative binding of eukaryotic initiation factor 4E to m⁷GpppN (cap structure) on the 5′ end of mRNA. A series of polyamines with varied numbers of aminoethylene repeats in their side chains were prepared by an aminolysis reaction of poly­(β-benzyl-l-aspartate) and paired with mRNA to form PICs. PICs formed from polyamines with higher numbers of aminoethylene repeats preserved the original translational efficiency to naked mRNA, whereas the efficiency significantly dropped by decreasing the number of aminoethylene repeats in the polyamines. Immunoprecipitation assays using anti-eIF4E antibodies revealed that the binding affinity of eIF4E to the cap structure of mRNA in the PIC was sensitive to the number of charged aminoethylene repeats in the polyamine side chain and was strongly correlated with their translational efficiency. These results indicate that the fine-tuning of the polyamine structure plays a critical role in maximizing the translational efficiency of mRNA in the PICs having potential utility as mRNA delivery vehicles

In vitro analysis of Toll-like receptor (TLR) signalling after mRNA introduction.

<p>To evaluate mRNA-mediated TLR signaling, HEK293 cells expressing TLR7 (grey bars) were used. HEK293 cells expressing TLR9 (white bars), which does not recognise mRNA, were also used as a negative control. Cells were treated with naked mRNA, polyplex nanomicelle or Lipofectamine 2000 using unmodified mRNA (a, b) or modified mRNA (c, d). Expression of interleukin (IL)-8 (a, c) and interferon (IFN)-β1 (b, d) was measured at transcriptional levels using real-time quantitative PCR (RT-PCR) 4 h after mRNA introduction. The data are presented as the mean ± standard error of the mean (s.e.m.) (N = 6). Statistical significance was assessed by 2-tailed Student's t-test, *, P<0.05, ***, P<0.001.</p