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.

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation.

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 µm diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

An injectable spheroid system with genetic modification for cell transplantation therapy.

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-µm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications.

Homo-catiomer integration into PEGylated polyplex micelle from block-catiomer for systemic anti-angiogenic gene therapy for fibrotic pancreatic tumors.

Homo-poly{N'-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide} [PAsp(DET), H] was attempted to integrate into poly (ethylene glycol) (PEG)-b-PAsp(DET)] (B) formulated polyplex micelle with the aim of enhancing cell transfection efficiency for PEGylated polyplex micelle via H integration. In vitro evaluations verified H integration of potent stimulation in enhancing cell-transfecting activity of PEGylated polyplex micelles via promoted cellular uptake and facilitated endosome escape. In vivo anti-angiogenic tumor suppression evaluations validated the feasibility of H integration in promoting gene transfection to the affected cells via systemic administration, where loaded anti-angiogenic gene remarkably expressed in the tumor site, thereby imparting significant inhibitory effect on the growth of vascular endothelial cells, ultimately leading to potent tumor growth suppression. These results demonstrated potency of H integration for enhanced transfection activity and potential usage in systemic applications, which could have important implications on the strategic use of H integration in the non-viral gene carrier design.

Gene transfection to spheroid culture system on micropatterned culture plate by polyplex nanomicelle: a novel platform of genetically-modified cell transplantation.

Three-dimensional (3D) cellular spheroids have attracted much attention as a transplantation procedure because the increased cell-to-cell interaction in spheroids enhances cell survival and its functions after the transplantation into the body. Furthermore, the potency of spheroidal cells may be further improved by introducing transgenes to augment cellular functions as well as enhance the paracrine effects by secreting key proteins involved in the essential cellular signaling cascades. In this study, we organized a new platform for genetically-modified cell transplantation by combining a microfabricated culture system for 3D spheroid formation with a newly developed non-viral transfection system, polyplex nanomicelle. After transfection of Gaussia luciferase using the nanomicelle, the prolonged luciferase expression was obtained for more than a month with continuous albumin secretion from the hepatocyte spheroids to the level comparable with control spheroids receiving no transfection. In contrast, by the transfection using FuGENE HD, a commercially available lipid-based reagent, the luciferase expression was obtained, yet the albumin secretion was significantly decreased with disintegration of the spheroid architecture. To assess the feasibility of the hepatocyte spheroids for in vivo transplantation, the spheroids were recovered by the use of micropatterned culture plate functionalized with thermosensitive polymer and dispersed into Matrigel(TM) Matrix. The luciferase expression as well as albumin secretion was maintained for more than a month from the spheroids in the Matrix. Thus, the combination of spheroid cell culture on micropatterned plates with gene introduction using polyplex nanomicelle is a promising platform for genetically-modified cell transplantation to achieve sustained transgene expression with maintaining innate cell functions.