Meike N. Leiske

Tailoring Cellular Uptake and Fluorescence of Poly(2-oxazoline)-based Nanogels.

Controlling the size and charge of nanometer-sized objects is of upmost importance for their interactions with cells. We herein present the synthesis of poly(2-oxazoline) based nanogels comprising a hydrophilic shell and an amine containing core compartment. Amine groups were cross-linked using glutaraldehyde resulting in imine based nanogels. As a drug model, amino fluorescein was covalently immobilized within the core, quenching excessive aldehyde functions. By varying the amount of cross-linker, the zeta potential and, hence, the cellular uptake could be adjusted. The fluorescence of the nanogels was found to be dependent on the cross-linking density. Finally, the hemocompatibility of the described systems was studied by hemolysis and erythrocyte aggregation assays. While cellular uptake was shown to be dependent on the zeta potential of the nanogel, no harmful effects to red blood cells was observed, rendering the present system as an interesting toolbox for the production of nanomaterials with a defined biological interaction profile.

Cationic ring-opening polymerization of protected oxazolidine imines resulting in gradient copolymers of poly(2-oxazoline) and poly(urea)

Poly(urea)s are a polymer class widely used in industry. Their utilization in biomedical applications is already described, however, the use of controlled polymerization methods instead of polycondensation approaches would allow a better control over the degree of polymerization and the dispersity of the resulting polymers, improving their suitability for this particular field of application. Cationic ring-opening polymerization (CROP) as a chain growth polymerization enables those requirements and, additionally, allows the copolymerization with 2-oxazolines, which are generally known for their biocompatibility. In this report, a Boc protected oxazolidine imine monomer is synthesized and polymerized in a homopolymerization, as well as in a copolymerization with 2-ethyl-2-oxazoline (EtOx) via CROP. The synthesized polymers were analyzed regarding their chemical and physical properties, using NMR, GC, MALDI-MS, SEC, TGA and DSC. Copolymerization kinetics revealed the formation of quasi-block copolymers, able to self-assemble in aqueous solution as indicated by DLS.

Mission ImPOxable – or the unknown utilization of non-toxic poly(2-oxazoline)s as cryoprotectants and surfactants at the same time

Polymer based nanoparticles offer great opportunities for diverse applications, i.e. their drug delivery potential is promising. However, their major drawback is identified in preparation via the nanoemulsion technique, which is needed for the encapsulation of hydrophilic drugs and whereby the utilization of surfactants, e.g. poly(vinyl alcohol) (PVA), is mandatory. Furthermore, the preparation of nanoparticles is critical due to the need of lyophilization for storage. For this reason it is common to use cryoprotectants, which are usually sugar based. In the current study, we present the use of non-toxic, water-soluble poly(2-oxazoline)s (P(Ox)s) in terms of polymeric nanoparticle stabilizers for preparation, purification, and lyophilization. The nanoparticles were characterized via dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryoTEM). The use of hydrophilic P(Ox)s with a degree of polymerization of about 60 yielded stable nanoparticles. For the preparation via nanoemulsion a PDI below 0.2 could be obtained after adjustment of the surfactant concentration. All nanoparticles were in the size range of 100 to 200 nm according to DLS. Furthermore, the addition of P(Ox) was beneficial during particle purification via centrifugation and filtration as well as lyophilization, yielding nanoparticles with a PDI below 0.3 as determined via DLS and confirmed via cryoTEM measurements. Cytotoxicity, hemolysis and erythrocyte aggregation measurements of these P(Ox)s did not show any harmful effect on the treated cells.

How To Tune the Gene Delivery and Biocompatibility of Poly(2-(4-aminobutyl)-2-oxazoline) by Self- and Coassembly

Despite their promising potential in gene transfection, the toxicity and limited efficiency of cationic polymers as nonviral vectors are major obstacles for their broader application. The large amount of cationic charges, for example, in poly(ethylene imine) (PEI) is known to be advantageous in terms of their transfection efficiency but goes hand-in-hand with a high toxicity. Consequently, an efficient shielding of the charges is required to minimize toxic effects. In this study, we use a simple mixed-micelle approach to optimize the required charge density for efficient DNA complex formation and to minimize toxicity by using a biocompatible polymer. In detail, we coassembled mixed poly(2-oxazoline) nanostructures ( d ≈ 100 nm) consisting of a hydrophobic-cationic block copolymer (P(NonOx52- b-AmOx184)) and a hydrophobic-hydrophilic stealth block copolymer (P(EtOx155- b-NonOx76) in ratios of 0, 20, 40, 60, 80, and 100 wt % P(NonOx52- b-AmOx184). All micelles with cationic polymers exhibited a very good DNA binding efficiency and dissociation ability, while the bio- and hemocompatibility improved with increasing EtOx content. Analytics via confocal laser scanning microscopy and flow cytometry showed an enhanced cellular uptake, transfection ability, and biocompatibility of all prepared micelleplexes compared to AmOx homopolymers. Micelleplexes with 80 or 100 wt % revealed a similar transfection efficiency as PEI, while the cell viability was significantly higher (80 to 90% compared to 60% for PEI).

Tumor targeting with pH-responsive poly(2-oxazoline)-based nanogels for metronomic doxorubicin treatment

The synthesis of a new nanogel drug carrier system loaded with the anti-cancer drug doxorubicin (DOX) is presented. Poly(2-oxazoline) (POx) based nanogels from block copolymer micelles were cross-linked and covalently loaded with DOX using pH-sensitive Schiff’ base chemistry. DOX loaded POx based nanogels showed a toxicity profile comparable to the free drug, while unloaded drug carriers showed no toxicity. Hemolytic activity and erythrocyte aggregation of the drug delivery system was found to be low and cellular uptake was investigated by flow cytometry and fluorescence microscopy. While the amount of internalized drug was enhanced when incorporated into a nanogel, the release of the drug into the nucleus was delayed. For in vivo investigations the nanogel drug delivery system was combined with a metronomic treatment of DOX. Low doses of free DOX were compared to equivalent DOX loaded nanogels in a xenograft mouse model. Treatment with POx based nanogels revealed a significant tumor growth inhibition and increase in survival time, while pure DOX alone had no effect on tumor progression. The biodistribution was investigated by microscopy of organs of mice and revealed a predominant localization of DOX within tumorous tissue. Thus, the POx based nanogel system revealed a therapeutic efficiency despite the low DOX concentrations and could be a promising strategy to control tumor growth with fewer side effects.

One-Pot Synthesis of Block Copolymers by a Combination of Living Cationic and Controlled Radical Polymerization

The reversible addition-fragmentation chain-transfer (RAFT) process represents a sophisticated polymerization technique for the preparation of tailored and well-defined polymers from acrylates, acrylamides, and (meth)acrylates. The direct switching from other methods, such as cationic polymerizations, without the need for tedious functionalization and purification steps remains challenging. Within this study, it is demonstrated that poly(2-oxazoline) (P(Ox)) macro chain-transfer agents (macro-CTAs) can be prepared through the quenching of the cationic ring-opening polymerization with a carbonotrithioate salt. The end-functionalization of the P(Ox)s is observed to be almost quantitative and the macro-CTAs could be directly used for RAFT polymerization without further purification. This one-pot procedure could be extended to a variety of (multi)block copolymers consisting of different 2-oxazolines and acrylates with good-to-excellent control. Kinetic studies revealed the controlled polymerization of block copolymers, which are further accessible for α- and ω-end-functionalization. The simplicity and versatility of the approach promise a straightforward access to block copolymers from cationic and controlled radical polymerizations.

Smart pH-sensitive Nanogels for Controlled Release in Acidic Environment

The encapsulation of therapeutic compounds into nanosized delivery vectors has become an important strategy to improve efficiency and reduce side effects in drug delivery applications. Here, we report the synthesis of pH-sensitive nanogels, which are based on the monomer N-[(2,2-dimethyl-1,3-dioxolane)methyl]acrylamide (DMDOMA) bearing an acid cleavable acetal group. Degradation studies revealed that these nanogels hydrolyze under acidic conditions and degrade completely, depending on the cross-linker, but are stable in physiological environment. The best performing system was further studied regarding its release kinetics using the anticancer drug doxorubicin. In vitro studies revealed a good compatibility of the unloaded nanogel and the capability of the doxorubicin loaded nanogel to mediate cytotoxic effects in a concentration and time-dependent manner with an even higher efficiency than the free drug. Based on the investigated features, the presented nanogels represent a promising and conveniently prepared alternative to existing carrier systems for drug delivery.