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

Dual-Responsive Magnetic Core–Shell Nanoparticles for Nonviral Gene Delivery and Cell Separation

We present the synthesis of dual-responsive (pH and temperature) magnetic core–shell nanoparticles utilizing the grafting-from approach. First, oleic acid stabilized superparamagnetic maghemite (γ-Fe₂O₃) nanoparticles (NPs), prepared by thermal decomposition of iron pentacarbonyl, were surface-functionalized with ATRP initiating sites bearing a dopamine anchor group via ligand exchange. Subsequently, 2-(dimethylamino)­ethyl methacrylate (DMAEMA) was polymerized from the surface by ATRP, yielding dual-responsive magnetic core–shell NPs (γ-Fe₂O₃@PDMAEMA). The attachment of the dopamine anchor group on the nanoparticle's surface is shown to be reversible to a certain extent, resulting in a grafting density of 0.15 chains per nm² after purification. Nevertheless, the grafted NPs show excellent long-term stability in water over a wide pH range and exhibit a pH- and temperature-dependent reversible agglomeration, as revealed by turbidimetry. The efficiency of γ-Fe₂O₃@PDMAEMA hybrid nanoparticles as a potential transfection agent was explored under standard conditions in CHO-K1 cells. Remarkably, γ-Fe₂O₃@PDMAEMA led to a 2-fold increase in the transfection efficiency without increasing the cytotoxicity, as compared to polyethyleneimine (PEI), and yielded on average more than 50% transfected cells. Moreover, after transfection with the hybrid nanoparticles, the cells acquired magnetic properties that could be used for selective isolation of transfected cells

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-<i>block</i>-poly(methacrylic acid)-<i>block</i>-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 <i>in vitro</i> 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

Star-Shaped Drug Carriers for Doxorubicin with POEGMA and POEtOxMA Brush-like Shells: A Structural, Physical, and Biological Comparison

The synthesis of amphiphilic star-shaped poly­(ε-caprolactone)-<i>block</i>-poly­(oligo­(ethylene glycol)­methacrylate)­s ([PCL₁₈-<i>b</i>-POEGMA]₄) and poly­(ε-caprolactone)-<i>block</i>-poly­(oligo­(2-ethyl-2-oxazoline)­methacrylate)­s ([PCL₁₈-<i>b</i>-POEtOxMA]₄) is presented. Unimolecular behavior in aqueous systems is observed with the tendency to form loose aggregates for both hydrophilic shell types. The comparison of OEGMA and OEtOxMA reveals that the molar mass of the macromonomer in the hydrophilic shell rather than the mere length is the crucial factor to form an efficiently stabilizing hydrophilic shell. A hydrophilic/lipophilic balance of 0.8 is shown to stabilize unimolecular micelles in water. An extensive in vitro biological evaluation shows neither blood nor cytotoxicity. The applicability of the polymers as drug delivery systems was proven by the encapsulation of the anticancer drug doxorubicin, whose cytotoxic effect was retarded in comparison to the free drug