A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo.

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07-07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07-07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07-07059 only marginally inhibits mitochondrial respiration and function. V07-07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07-07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)-diacetate N-succinimidyl ester, V07-07059 proved superior regarding toxicity and photostability.

Selective Uptake of a Fructose Glycopolymer Prepared by RAFT Polymerization into Human Breast Cancer Cells.

A new methacrylic fructose glycomonomer is synthesized and copolymerized with N-isopropyl acrylamide by reversible addition fragmentation chain transfer (RAFT) poly-merization. By additional copolymerization of the analog mannose, glucose, and galactose glycomonomers, a set of glycopolymers is obtained which vary in the type of sugar attached to the polyacrylamide backbone. The glycopolymers are subsequently deprotected and characterized by size exclusion chromatography, FT-IR and NMR spectroscopy, elemental analysis, as well as turbidimetry, revealing the thermoresponsive character of all synthesized glycopolymers. The deprotected glycopolymers are subsequently labeled with a Rhodamine B derivative, utilizing the thiol-functionalities derived from the RAFT endgroups. As concluded from the ArlamaBlue assay, the glycopolymers are not cytotoxic. Finally, cellular uptake studies reveal a higher uptake of the fructose polymer into MDA-MB-231 breast cancer cells compared to the other glycopolymers, which demonstrates the high potential of fructosylated polymers for potential applications in the targeted treatment of breast cancer.

Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo.

Polymer-based nanoparticles are promising drug delivery systems allowing the development of new drug and treatment strategies with reduced side effects. However, it remains a challenge to screen for new and effective nanoparticle-based systems in vitro. Important factors influencing the behavior of nanoparticles in vivo cannot be simulated in screening assays in vitro, which still represent the main tools in academic research and pharmaceutical industry. These systems have serious drawbacks in the development of nanoparticle-based drug delivery systems, since they do not consider the highly complex processes influencing nanoparticle clearance, distribution, and uptake in vivo. In particular, the transfer of in vitro nanoparticle performance to in vivo models often fails, demonstrating the urgent need for novel in vitro tools that can imitate aspects of the in vivo situation more accurate. Dynamic cell culture, where cells are cultured and incubated in the presence of shear stress has the potential to bridge this gap by mimicking key-features of organs and vessels. Our approach implements and compares a chip-based dynamic cell culture model to the common static cell culture and mouse model to assess its capability to predict the in vivo success more accurately, by using a well-defined poly((methyl methacrylate)-co-(methacrylic acid)) and poly((methyl methacrylate)-co-(2-dimethylamino ethylmethacrylate)) based nanoparticle library. After characterization in static and dynamic in vitro cell culture we were able to show that physiological conditions such as cell-cell communication of co-cultured endothelial cells and macrophages as well as mechanotransductive signaling through shear stress significantly alter cellular nanoparticle uptake. In addition, it could be demonstrated by using dynamic cell cultures that the in vivo situation is simulated more accurately and thereby can be applied as a novel system to investigate the performance of nanoparticle systems in vivo more reliable.

The influence of polymer architecture on in vitro pDNA transfection.

In 2012, the first gene therapy agent was approved by the Europe Medicines Agency leading to increased interest in this research field. Beside viruses, non-viral agents based on lipids or polymers represent aspiring alternatives to deliver the genetic material. Different hurdles have to be overcome depending on the kind of nucleic acid used, where plasmid DNA (pDNA) and small interfering RNA represent the common ones. The main challenge for transfection agents, in particular for pDNA delivery, is the transfer to the cell and into the cell nuclei. Within the group of transfection vesicles, cationic polymers show promising features and variability, as they can be synthesized with tailor-made physical and chemical properties (architectures and functionalisation). In the field of polymer-based gene delivery, the tuning potential of polymers by using different architectures like graft and star-shaped polymers as well as self-assembled block copolymers is immense. In particular, in the last few years numerous new polymer designs showed enhanced transfection properties in combination with good biocompatibility. Furthermore, new insights into the transfection mechanism demonstrated the continuous progress in this field. Polymer architecture influences the polyplex characteristics and the latter has an impact on the transfection mechanism, e.g. the interaction with the cellular membrane depends on the polyplex shape. Moreover, polyplex dissociation can be easily influenced by the polymer chemistry, thus biodegradable linkers lead to well suited polymers with reduced toxicity and high delivery potential, and are also promising for in vivo applications. This review focuses on the influence of polymer architectures for pDNA transfection in vitro, showing recent developments and insights. The theoretical background concerning the biological challenges for cationic polymers and the impact of graft- or star-shaped architectures as well as self-assembled structures will be presented in detail.

A cationic poly(2-oxazoline) with high in vitro transfection efficiency identified by a library approach.

To date, cationic polymers with high transfection efficiencies (TE) often have a high cytotoxicity. By screening an 18-membered library of cationic 2-oxazoline-based polymers, a polymer with similar TE as linear poly(ethylene imine) but no detectable cytotoxicity at the investigated concentrations could be identified. The influence of the polymer side chain hydrophobicity and the type and content of amino groups on the pDNA condensation, the TE, the cytotoxicity, the cellular membrane interaction as well as the size, charge, and stability of the polyplexes was studied. Primary amines and an amine content of at least 40% were required for an efficient TE. While polymers with short side chains were non-toxic up to an amine content of 40%, long hydrophobic side chains induced a high cytotoxicity.

Small but powerful: co-assembly of polyether-based triblock terpolymers into sub-30 nm micelles and synergistic effects on cellular interactions.

We introduce a versatile ABC triblock terpoly- mer platform based on poly(ethylene oxide)-block-poly(allyl glycidyl ether)-block-poly(tert-butyl glycidyl ether) (PEO-b-PAGE-b-PtBGE) and subsequent functionalization of the PAGE segment with thiogalactose (hydroxyl), cysteamine (amino), and 2-mercaptopropionic acid (carboxy) by thiol-ene chemistry. These materials are used to prepare core-shell-corona micelles with a PtBGE core, a PAGE shell, and a PEO corona and sizes below 30 nm in aqueous media. We investigate the influence of different functional groups on micelle formation and cellular uptake. Moreover, co-assembly of differently functionalized materials allows to create micelles with a mixed shell and adjustable charge and, in that way, important characteristics such as cell uptake or cytotoxicity can be controlled. Furthermore, we demonstrate that even the uptake mechanism depends on the substitution pattern of the underlying triblock terpolymer.

A paradigm change: efficient transfection of human leukemia cells by stimuli-responsive multicompartment micelles.

The controlled nonviral delivery of genetic material using cationic polymers into cells has been of interest during the past three decades, yet the ideal delivery agent featuring utmost transfection efficiency and low cytotoxicity still has to be developed. Here, we demonstrate that multicompartment micelles from stimuli-responsive triblock terpolymers, polybutadiene-block-poly(methacrylic acid)-block-poly(2-(dimethylamino)ethyl methacrylate) (BMAAD), are promising candidates. The structures exhibit a patchy shell, consisting of amphiphilic (interpolyelectrolyte complexes, MAA and D) and cationic patches (excess D), generating a surface reminiscent to those of certain viruses and capable of undergoing pH-dependent changes in charge stoichiometry. After polyplex formation with plasmid DNA, superior transfection efficiencies can be reached for both adherent cells and human leukemia cells. Compared to the gold standard PEI, remarkable improvements and a number of advantages were identified for this system, including increased cellular uptake and an improved release of the genetic material, accompanied by fast and efficient endosomal escape. Furthermore, high sedimentation rates might be beneficial regarding in vitro applications.

Parallel high-throughput screening of polymer vectors for nonviral gene delivery: evaluation of structure-property relationships of transfection.

In recent years, "high-throughput" (HT) has turned into a keyword in polymer research. In this study, we present a novel HT workflow for the investigation of cationic polymers for gene delivery applications. For this purpose, various poly(ethylene imine)s (PEI) were used as representative vectors and investigated via HT-assays in a 96-well plate format, starting from polyplex preparation up to the examination of the transfection process. In detail, automated polyplex preparation, complex size determination, DNA binding affinity, polyplex stability, cytotoxicity, and transfection efficiency were performed in the well plate format. With standard techniques, investigation of the biological properties of polymers is quite time-consuming, so only a limited number of materials and conditions (such as pH, buffer composition, and concentration) can be examined. The approach described here allows many different polymers and parameters to be tested for transfection properties and cytotoxicity, giving faster insights into structure-activity relationships for biological activity.