Amino-Acid-Derived Anionic Polyacrylamides with Tailored Hydrophobicity-Physicochemical Properties and Cellular Interactions.

Polyanions can internalize into cells via endocytosis without any cell disruption and are therefore interesting materials for biomedical applications. In this study, amino-acid-derived polyanions with different alkyl side-chains are synthesized via postpolymerization modification of poly(pentafluorophenyl acrylate), which is synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization, to obtain polyanions with tailored hydrophobicity and alkyl branching. The success of the reaction is verified by size-exclusion chromatography, NMR spectroscopy, and infrared spectroscopy. The hydrophobicity, surface charge, and pH dependence are investigated in detail by titrations, high-performance liquid chromatography, and partition coefficient measurements. Remarkably, the determined pK a-values for all synthesized polyanions are very similar to those of poly(acrylic acid) (pK a = 4.5), despite detectable differences in hydrophobicity. Interactions between amino-acid-derived polyanions with L929 fibroblasts reveal very slow cell association as well as accumulation of polymers in the cell membrane. Notably, the more hydrophobic amino-acid-derived polyanions show higher cell association. Our results emphasize the importance of macromolecular engineering toward ideal charge and hydrophobicity for polymer association with cell membranes and internalization. This study further highlights the potential of amino-acid-derived polymers and the diversity they provide for tailoring properties toward drug delivery applications.

Folic Acid-Conjugated Brush Polymers Show Enhanced Blood-Brain Barrier Crossing in Static and Dynamic In Vitro Models Toward Brain Cancer Targeting Therapy.

Over the past decades, evidence has consistently shown that treatment of central nervous system (CNS)-related disorders, including Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis, and brain cancer, is limited due to the presence of the blood-brain barrier (BBB). To assist with the development of new therapeutics, it is crucial to engineer a drug delivery system that can cross the BBB efficiently and reach target cells within the brain. In this study, we present a potentially efficient strategy for targeted brain delivery through utilization of folic acid (FA)-conjugated brush polymers, that specifically target the reduced folate carrier (RFC, SLC19A1) expressed on brain endothelial cells. Here, azide (N3)-decorated brush polymers were prepared in a straightforward manner coupling a heterotelechelic α-NH2, ω-N3-poly(2-ethyl-2-oxazoline) (NH2-PEtOx-N3) to N-acylated poly(amino ester) (NPAE)-based brushes. Strain-promoted azide-alkyne cycloaddition (SPAAC) 'click chemistry' with DBCO-folic acid (FA) yielded FA-brush polymers. Interestingly, while azide functionalization of the brush polymers dramatically reduced their association to brain microvascular endothelial cells (hCMEC/D3), the introduction of FA to azide led to a substantial accumulation of the brush polymers in hCMEC/D3 cells. The ability of the polymeric brush polymers to traverse the BBB was quantitatively assessed using different in vitro BBB models including static Transwell and microfluidic platforms. FA-brush polymers showed efficient transport across hCMEC/D3 cells in a manner dependent on FA composition, whereas nonfunctionalized brush polymers exhibited limited trafficking under the same conditions. Further, cellular uptake inhibition studies suggested that the interaction and transport pathway of FA-brush polymers across BBB relies on the RFC-mediated pathways. The potential application of the developed FA-brush polymers in brain cancer delivery was also investigated in a microfluidic model of BBB-glioblastoma. Brush polymers with more FA units successfully presented an enhanced accumulation into U-87 MG glioma cells following its BBB crossing, compared to controls. These results demonstrate that FA-modified brush polymers hold a great potential for more efficient delivery of future brain therapeutics.

Vitamin B3 Containing Polymers for Nanodelivery.

Polymeric nanoparticles (NPs) with an integrated dual delivery system enable the controlled release of bioactive molecules and drugs, providing therapeutic advantages. Key design targets include high biocompatibility, cellular uptake, and encapsulating efficiency. In this study, a polymer library derived from niacin, also known as vitamin B3 is synthesized. The library comprises poly(2-(acryloyloxy)ethyl nicotinate) (PAEN), poly(2-acrylamidoethyl nicotinate) (PAAEN), and poly(N-(2-acrylamidoethyl)nicotinamide) (PAAENA), with varying hydrophilicity in the backbone and pendant group linker. All polymers are formulated, and those with increased hydrophobicity yield NPs with homogeneous spherical distribution and diameters below 150 nm, as confirmed by scanning electron microscopy and dynamic light scattering. Encapsulation studies utilizing a model drug, neutral lipid orange (NLO), reveal the influence of polymer backbone on encapsulation efficiency. Specifically, efficiencies of 46% and 96% are observed with acrylate and acrylamide backbones, respectively. Biological investigations showed that P(AEN) and P(AAEN) NPs are non-toxic up to 300 µg mL-1, exhibit superior cellular uptake, and boost cell metabolic activity. The latter is attributed to the cellular release of niacin, a precursor to nicotinamide adenine dinucleotide (NAD), a central coenzyme in metabolism. The results underline the potential of nutrient-derived polymers as pro-nutrient and drug-delivery materials.

Comparison of the Anti-inflammatory Activity and Cellular Interaction of Brush Polymer-N-Acetyl Cysteine Conjugates in Human and Murine Microglial Cell Lines.

Microglia-mediated neuroinflammation is commonly associated with neurodegeneration and has been implicated in several neurological disorders, such as Alzheimer's disease and Parkinson's disease. Therefore, it is crucial to develop a detailed understanding of the interaction of potential nanocarriers with microglial cells to efficiently deliver anti-inflammatory molecules. In this study, we applied brush polymers as a modular platform to systematically investigate their association with murine (BV-2) and human (HMC3) microglial cell lines in the presence and absence of the pro-inflammatory inducer lipopolysaccharide (LPS) using flow cytometry. Brush polymers of different sizes and shapes, ranging from ellipsoid to worm-like cylinders, were prepared through a combination of the two building blocks carboxylated N-acylated poly(aminoester)s (NPAEs)-based polymers and poly(2-ethyl-2-oxazoline)-NH2 (PEtOx-NH2) and characterized by 1H NMR spectroscopy, size exclusion chromatography, and small-angle neutron scattering. Generally, ellipsoidal particles showed the highest cellular association. Moreover, while no significant differences in murine cell association were observed, the brush polymers revealed a significant accumulation in LPS-activated human microglia compared to resting cells, emphasizing their higher affinity to activated HMC3 cells. Brush polymers with the highest cell association were further modified with the anti-inflammatory agent N-acetyl cysteine (NAC) in a reversible manner. The brush polymer-NAC conjugates were found to significantly attenuate the production of interleukin 6 (p < 0.001) in LPS-activated HMC3 cells compared to LPS-activated BV-2 cells. Thus, the presented brush polymer-NAC conjugates showed a high anti-inflammatory activity in human microglia, suggesting their potential for disease-targeted therapy of microglial-mediated neuroinflammation in the future.

Impact of the polymer backbone chemistry on interactions of amino-acid-derived zwitterionic polymers with cells.

Zwitterionic polymers are known to interact with cells and have been shown to reveal cancer cell specificity. In this work, the importance of the chemistry of the polymer backbone for the cellular specificity of amino-acid-derived polyzwitterions is demonstrated. A series of glutamic acid (Glu)-based vinyl monomers (i.e., an acrylate, a methacrylate, an acrylamide, and a methacrylamide) were prepared and used for reversible addition-fragmentation chain-transfer (RAFT) polymerisation, yielding defined polymers with narrow size distribution (Р< 1.3). All Glu-functionalised, zwitterionic polymers revealed high cytocompatibility; however, differences in cellular association and specificity were observed. In particular, the methacrylamide-derived polymers showed high association with both, breast cancer cells and non-cancerous dendritic cells and, consequently, lack specificity. In contrast, high specificity to only breast cancer cells was observed for polyacrylates, -methacrylates, and -acrylamides. Detailed analysis of the polymers revealed differences in hydrophobicity, zeta potential, and potential side chain hydrolysis, which are impacted by the polymer backbone and might be responsible for the altered the cell association of these polymers. It is shown that a slightly negative net charge is preferred over a neutral charge to retain cell specificity. This was also confirmed by association experiments in the presence of competitive amino acid transporter substrates. The affinity of slightly negatively charged Glu-derived polymers to the xCT Glu/cystine cell membrane antiporter was found to be higher than that of neutrally charged polymers. Our results emphasise the importance of the polymer backbone for the design of cell-specific polymers. This study further highlights the potential to tailor amino-acid-derived zwitterionic materials beyond their side chain functionality.

Accelerated Post-Polymerization Amidation of Polymers with Side-Chain Ester Groups by Intramolecular Activation.

The catalytic conversion of esters to amides represents new opportunities in the synthetic diversification and upcycling of polymers, as esters are commonly featured in various polymer structures. Yet, direct amidation is typically hampered by poor reaction kinetics and the effects of polymer structure on the reactivity remain poorly understood. We report the accelerated amidation for amines with additional hydrogen bond donating or accepting groups. These amines facilitate the expeditious (co)amidation of polymers with pendant ester groups, displaying at least a 400-fold higher reactivity relative to polyesters with esters in the main chain. Furthermore, a positive correlation between the reactivity and degree of polymerization for poly(methyl acrylate) suggests a hydrogen-bond mediated intramolecular activation of the esters, which was confirmed by FT-IR spectroscopy and basic molecular mechanics modeling. The reported method paves the way to synthesize diverse (co)polymers with amide side chains from readily available polymeric precursors.

Zwitterionic Amino Acid-Derived Polyacrylates as Smart Materials Exhibiting Cellular Specificity and Therapeutic Activity.

The synthesis of new amino acid-containing, cell-specific, therapeutically active polymers is presented. Amino acids served as starting material for the preparation of tailored polymers with different amino acids in the side chain. The reversible addition-fragmentation chain-transfer (RAFT) polymerization of acrylate monomers yielded polymers of narrow size distribution (D ≤ 1.3). In particular, glutamate (Glu)-functionalized, zwitterionic polymers revealed a high degree of cytocompatibility and cellular specificity, i.e., showing association to different cancer cell lines, but not with nontumor fibroblasts. Energy-dependent uptake mechanisms were confirmed by means of temperature-dependent cellular uptake experiments as well as localization of the polymers in cellular lysosomes determined by confocal laser scanning microscopy (CLSM). The amino acid receptor antagonist O-benzyl-l-serine (BzlSer) was chosen as an active ingredient for the design of therapeutic copolymers. RAFT copolymerization of Glu acrylate and BzlSer acrylate resulted in tailored macromolecules with distinct monomer ratios. The targeted, cytotoxic activity of copolymers was demonstrated by means of multiday in vitro cell viability assays. To this end, polymers with 25 mol % BzlSer content showed cytotoxicity against cancer cells, while leaving fibroblasts unaffected over a period of 3 days. Our results emphasize the importance of biologically derived materials to be included in synthetic polymers and the potential of zwitterionic, amino acid-derived materials for cellular targeting. Furthermore, it highlights that the fine balance between cellular specificity and unspecific cytotoxicity can be tailored by monomer ratios within a copolymer.

A Guideline for the Synthesis of Amino-Acid-Functionalized Monomers and Their Polymerizations.

Amino acids have emerged as a sustainable source for the design of functional polymers. Besides their wide availability, especially their high degree of biocompatibility makes them appealing for a broad range of applications in the biomedical research field. In addition to these favorable characteristics, the versatility of reactive functional groups in amino acids (i.e., carboxylic acids, amines, thiols, and hydroxyl groups) makes them suitable starting materials for various polymerization approaches, which include step- and chain-growth reactions. This review aims to provide an overview of strategies to incorporate amino acids into polymers. To this end, it focuses on the preparation of polymerizable monomers from amino acids, which yield main chain or side chain-functionalized polymers. Furthermore, postpolymerization modification approaches for polymer side chain functionalization are discussed. Amino acids are presented as a versatile platform for the development of polymers with tailored properties.

Nitrile-Functionalized Poly(2-oxazoline)s as a Versatile Platform for the Development of Polymer Therapeutics.

In recent years, polymers bearing reactive groups have received significant interest for biomedical applications. Numerous functional polymer platforms have been introduced, which allow for the preparation of materials with tailored properties via post-polymerization modifications. However, because of their reactivity, many functional groups are not compatible with the initial polymerization. The nitrile group is a highly interesting and relatively inert functionality that has mainly received attention in radical polymerizations. In this Article, a nitrile-functionalized 2-oxazoline monomer (2-(4-nitrile-butyl)-2-oxazoline, BuNiOx) is introduced, and its compatibility with the cationic ring-opening polymerization is demonstrated. Subsequently, the versatility of nitrile-functionalized poly(2-oxazoline)s (POx) is presented. To this end, diverse (co)polymers are synthesized and characterized by nuclear resonance spectroscopy, size-exclusion chromatography, and mass spectrometry. Amphiphilic block copolymers are shown to efficiently encapsulate the hydrophobic drug curcumin (CUR) in aqueous solution, and the anti-inflammatory effect of the CUR-containing nanostructures is presented in BV-2 microglia. Furthermore, the availability of the BuNiOx repeating units for post-polymerization modifications with hydroxylamine to yield amidoxime (AO)-functionalized POx is demonstrated. These AO-containing POx were successfully applied for the complexation of Fe(III) in a quantitative manner. In addition, AO-functionalized POx were shown to release nitric oxide intracellularly in BV-2 microglia. Thus nitrile-functionalized POx represent a promising and robust platform for the design of polymer therapeutics for a wide range of applications.

Interactions of core cross-linked poly(2-oxazoline) and poly(2-oxazine) micelles with immune cells in human blood.

Water-soluble poly(cyclic imino ether)s (PCIEs) have emerged as promising biocompatible polymers for nanomedicine applications in recent years. Despite their generally accepted stealth properties, there has been no comprehensive evaluation of their interactions with primary immune cells in human blood. Here we present a library of core cross-linked micelles (CCMs) containing various PCIE shells. Well-defined high molar mass CCMs (Mn > 175 kDa, Р< 1.2) of similar diameter (~20 nm) were synthesised using a cationic ring-opening polymerisation (CROP) - surfactant-free reversible addition-fragmentation chain-transfer (RAFT) emulsion polymerisation strategy. The stealth properties of the different PCIE CCMs were assessed employing a whole human blood assay simulating the complex blood environment. Cell association studies revealed lower associations of poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) CCMs with blood immune cells compared to the respective poly(2-oxazine) (POz) CCMs. Noteworthy, PMeOx CCMs outperformed all other reported CCMs, showing overall low associations and only negligible differences in the presence and absence of serum proteins. This study highlights the importance of investigating individual nanomaterials under physiologically relevant conditions and further strengthens the position of PMeOx as a highly promising stealth material for biomedical applications.

Engineering Fluorescent Gold Nanoclusters Using Xanthate-Functionalized Hydrophilic Polymers: Toward Enhanced Monodispersity and Stability.

We introduce xanthate-functionalized poly(cyclic imino ethers)s (PCIEs), specifically poly(2-ethyl-2-oxazoline) and poly(2-ethyl-2-oxazine) given their stealth characteristics, as an attractive alternative to conventional thiol-based ligands for the synthesis of highly monodisperse and fluorescent gold nanoclusters (AuNCs). The xanthate in the PCIEs interacts with Au ions, acting as a well-controlled template for the direct formation of PCIE-AuNCs. This method yields red-emitting AuNCs with a narrow emission peak (λem = 645 nm), good quantum yield (4.3-4.8%), long fluorescence decay time (∼722-844 ns), and unprecedented product yield (>98%). The PCIE-AuNCs exhibit long-term colloidal stability, biocompatibility, and antifouling properties, enabling a prolonged blood circulation, lower nonspecific accumulation in major organs, and better renal clearance when compared with AuNCs without polymer coating. The advances made here in the synthesis of metal nanoclusters using xanthate-functionalized PCIEs could propel the production of highly monodisperse, biocompatible, and renally clearable nanoprobes in large-scale for different theranostic applications.

Utilization of 4-(trifluoromethyl)benzenesulfonates as Counter Ions Tunes the Initiator Efficiency of Sophisticated Initiators for the Preparation of Well-Defined poly(2-oxazoline)s.

During the last decades, poly(2-oxazoline)s (POx) have gained increased interest due to their versatility. In particular, cationic ring-opening polymerization (CROP) enables the synthesis of well-defined polymers bearing quantitative α- and ω-functionalities. In contrast to small initiating groups, the introduction of more sophisticated, respectively demanding groups remains challenging. To fulfill this challenge, the initiator should comply with one major requirement in order to yield well-defined polymers: a fast and complete initiation. The straight forward two-step synthesis of a novel initiator containing a 4-(trifluoromethyl)benzenesulfonate (fluorylate, TosCF3 ) counter-ion is herein presented to accomplish the introduction of a sophisticated functional 3-(2-(2-ethoxy)ethoxy)ethoxy)prop-1-ene (TEG) initiating group. Kinetic studies are conducted in acetonitrile and chlorobenzene using the hydrophilic 2-ethyl-2-oxazoline (EtOx) as well as the hydrophobic 2-octyl-2-oxazoline (OctOx) as monomers to examine the influences of the solvent as well as the different monomers. In particular, the initiator efficiency is determined by 1 H and 19 F nuclear magnetic resonance spectroscopy and compared to the corresponding tosylate (TEGTos) and triflate (TEGTf). It is shown that the fluorylate combines the stability of the tosylate and an enhanced propagation rate comparable to the triflate.

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 an 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.

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.

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).

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.

Site-Specific POxylation of Interleukin-4.

Polymer conjugated biologics form a multibillion dollar market, dominated by poly(ethylene glycol) (PEG). Recent reports linked PEGs to immunological concerns, fueling the need for alternative polymers. Therefore, we are presenting a strategy replacing PEG by poly(2-oxazoline) (POx) polymers using genetically engineered interleukin-4 (IL-4) featuring an unnatural amino acid for site-specific conjugation through bioorthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). Conjugation yields of IL-4-PEG were poor and did not respond to an increase in the copper catalyst. In contrast, POxylated IL-4 conjugates resulted in homogeneous conjugate outcome, as demonstrated electrophoretically by size exclusion chromatography and analytical ultracentrifugation. Furthermore, POxylation did not impair thermal and chemical stability, and preserved wild-type IL-4 activity for the conjugates as demonstrated by TF-1 cell proliferation and STAT-6 phosphorylation in HEK293T cells, respectively. In conclusion, POxylation provides an interesting alternative to PEGylation with superior outcome for the synthesis yield by CuAAC and resulting in conjugates with excellent thermal and chemical stress profiles as well as biological performances.

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.