Christopher V. Synatschke

Influence of Polymer Architecture and Molecular Weight of Poly(2-(dimethylamino)ethyl methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.

Nondestructive Light-Initiated Tuning of Layer-by-Layer Microcapsule Permeability

A nondestructive way to achieve remote, reversible, light-controlled tunable permeability of ultrathin shell microcapsules is demonstrated in this study. Microcapsules based on poly{[2-(methacryloyloxy)ethyl] trimethylammonium iodide} (PMETAI) star polyelectrolyte and poly(sodium 4-styrenesulfonate) (PSS) were prepared by a layer-by-layer (LbL) technique. We demonstrated stable microcapsules with controlled permeability with the arm number of a star polymer having significant effect on the assembly structure: the PMETAI star with 18 arms shows a more uniform and compact assembly structure. We observed that in contrast to regular microcapsules from linear polymers, the permeability of the star polymer microcapsules could be dramatically altered by photoinduced transformation of the trivalent hexacyanocobaltate ions into a mixture of mono- and divalent ions by using UV irradiation. The reversible contraction of PMETAI star polyelectrolyte arms and the compaction of star polyelectrolytes in the presence of multivalent counterions are considered to cause the dramatic photoinduced changes in microcapsule properties observed here. Remarkably, unlike the current mostly destructive approaches, the light-induced changes in microcapsule permeability are completely reversible and can be used for light-mediated loading/unloading control of microcapsules.

Nanoparticulate Nonviral Agent for the Effective Delivery of pDNA and siRNA to Differentiated Cells and Primary Human T Lymphocytes

Delivery of polynucleotides such as plasmid DNA (pDNA) and siRNA to nondividing and primary cells by nonviral vectors presents a considerable challenge. In this contribution, we introduce a novel type of PDMAEMA-based star-shaped nanoparticles that (i) are efficient transfection agents in clinically relevant and difficult-to-transfect human cells (Jurkat T cells, primary T lymphocytes) and (ii) can efficiently deliver siRNA to human primary T lymphocytes resulting to more than 40% silencing of the targeted gene. Transfection efficiencies achieved by the new vectors in serum-free medium are generally high and only slightly reduced in the presence of serum, while cytotoxicity and cell membrane disruptive potential at physiological pH are low. Therefore, these novel agents are expected to be promising carriers for nonviral gene transfer. Moreover, we propose a general design principle for the construction of polycationic nanoparticles capable of delivering nucleic acids to the above-mentioned cells.

Multicompartment micelles with adjustable poly(ethylene glycol) shell for efficient in vivo photodynamic therapy

We describe the preparation of well-defined multicompartment micelles from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers and their use as advanced drug delivery systems for photodynamic therapy (PDT). A porphyrazine derivative was incorporated into the hydrophobic core during self-assembly and served as a model drug and fluorescent probe at the same time. The initial micellar corona is formed by negatively charged PMAA and could be gradually changed to poly(ethylene glycol) (PEG) in a controlled fashion through interpolyelectrolyte complex formation of PMAA with positively charged poly(ethylene glycol)-block-poly(L-lysine) (PLL-b-PEG) diblock copolymers. At high degrees of PEGylation, a compartmentalized micellar corona was observed, with a stable bottlebrush-on-sphere morphology as demonstrated by cryo-TEM measurements. By in vitro cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their in vitro PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the in vivo blood circulation and tumor accumulation was found. Fully PEGylated micelles were detected for up to 24 h in the bloodstream and accumulated in solid subcutaneous A549 tumors, while non- or only partially PEGylated micelles were rapidly cleared and did not accumulate in tumor tissue. Efficient tumor growth suppression was shown for fully PEGylated micelles up to 20 days, demonstrating PDT efficacy in vivo.

Double-layered micellar interpolyelectrolyte complexes—how many shells to a core?

We report on the formation of double-layered micellar interpolyelectrolyte complexes (IPECs) from ABC triblock terpolymer precursor micelles and hydrophilic homo- or block copolymers. Polybutadiene-block-poly(1-methyl-2-vinyl pyridinium)-block-poly(sodium methacrylate) (PB-b-P2VPq-b-PMANa) block terpolymers form micelles in aqueous solution at high pH exhibiting a PB core, a P2VPq/PMANa intramicellar IPEC (im-IPEC) shell, and a PMANa corona, which is negatively charged. Upon mixing with either positively charged, quaternized poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMAq) homopolymers or its double-hydrophilic block copolymer with poly(ethylene oxide) (PEO-b-PDMAEMAq), a further IPEC shell is formed, rendering core–shell–shell–corona aggregates. The effects of the ratio of positive to negative charges, Z+/−, the composition of the block terpolymer micelles, and the length of the added Dq block were investigated. We show that within a certain Z+/− regime stable complex micellar IPECs featuring two distinguishable IPEC shells are formed. The so-formed complex particles were analyzed by dynamic light scattering and cryogenic transmission electron microscopy.

Hidden Structural Features of Multicompartment Micelles Revealed by Cryogenic Transmission Electron Tomography

The demand for ever more complex nanostructures in materials and soft matter nanoscience also requires sophisticated characterization tools for reliable visualization and interpretation of internal morphological features. Here, we address both aspects and present synthetic concepts for the compartmentalization of nanoparticle peripheries as well as their in situ tomographic characterization. We first form negatively charged spherical multicompartment micelles from ampholytic triblock terpolymers in aqueous media, followed by interpolyelectrolyte complex (IPEC) formation of the anionic corona with bis-hydrophilic cationic/neutral diblock copolymers. At a 1:1 stoichiometric ratio of anionic and cationic charges, the so-formed IPECs are charge neutral and thus phase separate from solution (water). The high chain density of the ionic grafts provides steric stabilization through the neutral PEO corona of the grafted diblock copolymer and suppresses collapse of the IPEC; instead, the dense grafting results in defined nanodomains oriented perpendicular to the micellar core. We analyze the 3D arrangements of the complex and purely organic compartments, in situ, by means of cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET). We study the effect of block lengths of the cationic and nonionic block on IPEC morphology, and while 2D cryo-TEM projections suggest similar morphologies, cryo-ET and computational 3D reconstruction reveal otherwise hidden structural features, e.g., planar IPEC brushes emanating from the micellar core.

DNA Melting Temperature Assay for Assessing the Stability of DNA Polyplexes Intended for Nonviral Gene Delivery

Many synthetic polycations have the ability to form complexes with the polyanion DNA, yet only a few, most notably poly(ethylene imine) (PEI), are efficient gene-delivery vehicles. Although a common explanation of this observation relies on the buffering capacity of the polycation, the intracellular stability of the complex may also play a role and should not be neglected. Assays typically used to follow complex formation, however, often do not provide the required information on stability. In this article, we propose the change in the DNA melting temperature observable after complex formation to be a significant indicator of complex stability. For a given DNA/polycation ratio, changes in the melting temperature are shown to depend on the polycation chemistry but not on the DNA topology or the polycation architecture. Effects of changes in the DNA/polycation ratio as well as the effect of polycation quaternization can be interpreted using the melting temperature assay. Finally, the assay was used to follow the displacement of DNA from the complexes by poly(methacrylic acid) or short single-stranded DNA sequences as competing polyanions.

Oligomeric dual functional antibacterial polycaprolactone

Dual functional antibacterial and biodegradable caprolactone (CL) based oligomers were prepared by ring-opening polymerization of caprolactone using a polyguanidine macroinitiator. Primary amino (–NH2) end-groups of linear poly(hexamethylene guanidine) hydrochloride (PHMG) acted as initiating sites for caprolactone polymerization leading to block copolymers which combine the antibacterial properties of PHMG with degradability provided by PCL. The block structure of the material was confirmed with 2D NMR and MALDI-ToF-MS. Furthermore, the material exhibited temperature dependent solubility (upper critical solution temperature) in polar solvents such as methanol. The oligomers showed high antibacterial activity (reduction of bacterial cells was more than 3 orders of magnitude) even at short incubation times depending on the concentration and PHMG : PCL ratio while maintaining enzymatic degradability and biocompatibility.

Co-assemblies of micelle-forming diblock copolymers and enzymes on graphite substrate for an improved design of biosensor systems

The adsorption of ionic amphiphilic diblock copolymers comprising a polycationic block, polybutadiene-block-poly(2-(dimethylamino)ethyl methacrylate) (PB-b-PDMAEMA); and its quaternized derivative (PB-b-PDMAEMAq) from aqueous media onto graphite-based surfaces was examined. Both diblock copolymers in aqueous solution form star-like micelles with a hydrophobic PB core and a cationic corona built up from either strong cationic PDMAEMAq or pH-sensitive PDMAEMA. AFM experiments show that PB-b-PDMAEMAq micelles interact slightly with a graphite surface providing films with a low surface coverage. PB-b-PDMAEMA micelles adsorbed onto a graphite surface at pH ≥ 7 result in a more homogeneous coverage of the graphite surface by the diblock copolymer. The adsorption of two enzymes, tyrosinase (Tyr) and choline oxidase (ChO) on the graphite surface premodified with these diblock copolymers was also monitored by AFM and by electrochemical measurements of the enzymatic activities of PB-b-PDMAEMA–Tyr and PB-b-PDMAEMA–ChO films. A pronounced increase in the enzymatic activity of tyrosinase was observed with the increasing concentration of PB-b-PDMAEMA micelles in solution used for their depositions. Also, a pronounced increase in the enzymatic activities of both tyrosinase and choline oxidase was observed with the increasing pH of the deposition of the micelles from 2 to 10. The enzymatic activity increases with the coverage of the graphite surface with the preadsorbed copolymer. Finally, the polymer–enzyme films were tested as biosensors for phenol (when tyrosinase was adsorbed) and choline (when choline oxidase was adsorbed) and their activity and stability were compared to already existing setups.

Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

Genetic modification (“transfection”) of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino) ethyl methacrylate (DMAEMA) building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293) and non-adherent (Jurkat, primary human T lymphocytes) mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities) had been grown from a center silsesquioxane or silica-coated γ-Fe2O3-core and one micellar structure self-assembled from poly(1,2-butadiene)-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate), provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution), but also the concentrations adjusted during polyplex formation, affected the results.