Tuning the Surface of Nanoparticles: Impact of Poly(2‐ethyl‐2‐oxazoline) on Protein Adsorption in Serum and Cellular Uptake

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2‐ethyl‐2‐oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non‐coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi‐angle dynamic light scattering, asymmetrical flow field‐flow fractionation, gel electrophoresis, and liquid chromatography‐mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non‐specific cellular uptake, particularly by macrophage‐like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

     

Phase‐transition characteristics of amphiphilic poly(2‐ethyl‐2‐oxazoline)/poly(ε‐caprolactone) block copolymers in aqueous solutions

Amphiphilic diblock and triblock copolymers of various block compositions based on hydrophilic poly(2‐ethyl‐2‐oxazoline) (PEtOz) and hydrophobic poly(ε‐caprolactone) were synthesized. The micelle formation of these block copolymers in aqueous media was confirmed by a fluorescence technique and dynamic light scattering. The critical micelle concentrations ranged from 35.5 to 4.6 mg/L for diblock copolymers and 4.7 to 9.0 mg/L for triblock copolymers, depending on the block composition. The phase‐transition behaviors of the block copolymers in concentrated aqueous solutions were investigated. When the temperature was increased, aqueous solutions of diblock and triblock copolymers exhibited gel–sol transition and precipitation, both of which were thermally reversible. The gel–sol transition‐ and precipitation temperatures were manipulated by adjustment of the block composition. As the hydrophobic portion of block copolymers became higher, a larger gel region was generated. In the presence of sodium chloride, the phase transitions were shifted to a lower temperature level. Sodium thiocyanate displaced the gel region and precipitation temperatures to a higher temperature level. The low molecular weight saccharides, such as glucose and maltose, contributed to the shift of phase‐transition temperatures to a lower temperature level, where glucose was more effective than maltose in lowering the gel–sol transition temperatures. The malonic acid that formed hydrogen bonds with the PEtOz shell of micelles was effective in lowering phase‐transition temperatures to 1.0M, above which concentration the block copolymer solutions formed complex precipitates. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 2400–2408, 2000     

Aqueous solution behavior of comb‐shaped poly(2‐ethyl‐2‐oxazoline)

A series of comb polymers consisting of a methacrylate backbone and poly(2‐ethyl‐2‐oxazoline) (PEtOx) side chains was synthesized by a combination of cationic ring‐opening polymerization and reversible addition–fragmentation chain transfer polymerization. Small‐angle neutron scattering (SANS) studies revealed a transition from an ellipsoidal to a cylindrical conformation in D₂O around a backbone degree of polymerization of 30. Comb‐shaped PEtOx has lowered T_g values but a similar elution behavior in liquid chromatography under critical conditions in comparison to its linear analog was observed. The lower critical solution temperature behavior of the polymers was investigated by turbidimetry, dynamic light scattering, transmission electron microscopy, and SANS revealing decreasing T_cp in aqueous solution with increasing molar mass, the presence of very few aggregated structures below T_cp, a contraction of the macromolecules at temperatures 5 °C above T_cp but no severe conformational change of the cylindrical structure. In addition, the phase diagram including cloud point and coexistence curve was developed showing an LCST of 75 °C of the binary mixture poly[oligo(2‐ethyl‐2‐oxazoline)methacrylate]/water. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

In vitro hemocompatibility and cytotoxicity study of poly(2‐methyl‐2‐oxazoline) for biomedical applications

Since poly(2‐methyl‐2‐oxazolines) (PMeOx) attract high attention for the potential use in drug delivery, cytotoxicity, and hemocompatibility of a set of PMeOxs with molar masses in the range from 2 to 20 kDa are systematically investigated under standardized conditions in terms of molar mass, concentration and time dependency. PMeOx polymers are well tolerated in red blood cell aggregation and hemolysis assays without any damaging effects even at high concentrations up to 80 mg/mL. Only in long term cytotoxicity tests PMeOx polymers moderately influence cell viability in a time, concentration, and molar mass dependent manner. Referring to these results it can be concluded that PEtOx could be promising nonionic hydrophilic polymers for many biomedical applications without any cyto‐ and hemotoxic effects at typically used therapeutic doses. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Comparative studies on miscibility and phase behavior of linear and star poly(2‐methyl‐2‐oxazoline) blends with poly(vinylidene fluoride)

The comparative studies on the miscibility and phase behavior between the blends of linear and star‐shaped poly(2‐methyl‐2‐oxazoline) with poly(vinylidene fluoride) (PVDF) were carried out in this work. The linear poly(2‐methyl‐2‐oxazoline) was synthesized by the ring opening polymerization of 2‐methyl‐2‐oxazoline in the presence of methyl p‐toluenesulfonate (MeOTs) whereas the star‐shaped poly(2‐methyl‐2‐oxazoline) was synthesized with octa(3‐iodopropyl) polyhedral oligomeric silsesquioxane [(IC₃H₆)₈Si₈O₁₂, OipPOSS] as an octafunctional initiator. The polymers with different topological structures were characterized by means of Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy. It is found that the star‐shaped poly(2‐methyl‐2‐oxazoline) was miscible with poly(vinylidene fluoride) (PVDF), which was evidenced by single glass‐transition temperature behavior and the equilibrium melting‐point depression. Nonetheless, the blends of linear poly(2‐methyl‐2‐oxazoline) with PVDF were phase‐separated. The difference in miscibility was ascribed to the topological effect of PMOx macromolecules on the miscibility. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 942–952, 2006     

Uni‐molecular nanoparticles of poly(2‐oxazoline) showing tunable thermoresponsive behaviors

Herein, cylindrical molecular bottlebrushes grafted with poly(2‐oxazoline) (POx) as a shaped tunable uni‐molecular nanoparticle were synthesized via the grafting‐onto approach. First, poly(glycidyl methacrylate) (PGMA) backbones with azide pendant units were prepared via reversible addition fragmentation transfer (RAFT) polymerization followed by post‐modification. The degree of polymerization (DP) of the backbones was tuned in a range from 20 to 800. Alkynyl‐terminated POx side chains were synthesized by living cationic ring opening polymerization (LCROP) of 2‐ethyl‐2‐oxazoline (EtOx) and 2‐methyl‐2‐oxazoline (MeOx), respectively. The DP of side chains was varied between 20 and 100. Then, the copper‐catalyzed azide‐alkynyl cycloaddition (CuAAC) click chemistry was conducted with a feed ratio of [alkynyl]:[azide] = 1.2:1 to yield a series of brushes. Depending on the DP of side chains, the grafting density ranged between 47 and 85%. The resulting brushlike nanoparticles exhibited shapes of sphere, rod and worm. Aqueous solutions of PEtOx brushes demonstrated a thermoresponsive behavior as a function of the length of backbones and side chains. Surprisingly, it was found that the lower critical solution temperature of PEtOx brushes increased with a length increase of backbones. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 174–183     

Tuning the LCST of poly(2‐cyclopropyl‐2‐oxazoline) via gradient copolymerization with 2‐ethyl‐2‐oxazoline

Poly(2‐propyl‐oxazoline)s can be prepared by living cationic ring‐opening polymerization of 2‐oxazolines and represent an emerging class of biocompatible polymers exhibiting a lower critical solution temperature in aqueous solution close to body temperature. However, their usability is limited by the irreversibility of the transition due to isothermal crystallization in case of poly(2‐isopropyl‐2‐oxazoline) and the rather low glass transition temperatures (T_g < 45 °C) of poly(2‐n‐propyl‐2‐oxazoline)‐based polymers. The copolymerization of 2‐cyclopropyl‐2‐oxazoline and 2‐ethyl‐2‐oxazoline presented herein yields gradient copolymers whose cloud point temperatures can be accurately tuned over a broad temperature range by simple variation of the composition. Surprisingly, all copolymers reveal lower T_gs than the corresponding homopolymers ascribed to suppression of interchain interactions. However, it is noteworthy that the copolymers still have T_gs > 45 °C, enabling convenient storage in the fridge for future biomedical formulations. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 3118–3122     

Microphase segregation and selective chain scission of poly(2‐methyl‐2‐oxazoline)‐block‐polystyrene

Herein, we report the design and synthesis of a block copolymer (BCP) with a high Flory–Huggins interaction parameter to access 10 nm feature sizes for potential lithographic applications. The investigated BCP is poly[(2‐methyl‐2‐oxazoline)‐block‐styrene] (PMeOx‐b‐PS), where the PMeOx segment functions as a hydrophilic segment. Two BCPs with different molecular weights were prepared using PMeOx as macroinitiator for copper(0) mediated controlled radical polymerization. The thin film self‐assembly of the obtained PMeOx‐b‐PS was performed by solvent annealing and investigated by atomic force microscopy. Both polymers formed PMeOx cylinders in a PS matrix with an average cylinder diameter of 10.5 nm. Additionally, the ability of the PMeOx domains to selectively degrade under ultraviolet irradiation was explored. It was shown that scission of the PMeOx block does occur selectively, and furthermore that the degraded domains can be removed while leaving the PS matrix intact. By combining synthetic accessibility, small feature sizes, and a selectively cleavable domain, this new BCP system holds significant promise as a lithographic mask for patterning surfaces with high precision. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 1349–1357     

Poly(2‐oxazoline) hydrogels crosslinked with aliphatic bis(2‐oxazoline)s: Properties,cytotoxicity, and cell cultivation

A series of hydrogels from 2‐ethyl‐2‐oxazoline and three bis(2‐oxazoline) crosslinkers—1,4‐butylene‐2,2′‐bis(2‐oxazoline), 1,6‐hexamethylene‐2,2′‐bis(2‐oxazoline), and 1,8‐octamethylene‐2,2′‐bis(2‐oxazoline)—are prepared. The hydrogels differ by the length of aliphatic chain of crosslinker and by the percentage of crosslinker (2–10%). The influence of the type and the percentage of the crosslinker on swelling properties, mechanical properties, and state of water is studied. The equilibrium swelling degree in water ranges from 2 to 20. With a proper selection of the crosslinker, Young's modulus can be varied from 10 kPa to almost 100 kPa. To evaluate the potential for medical applications, the cytotoxicity of extracts and the contact toxicity toward murine fibroblasts are measured. The hydrogels with the crosslinker containing a shorter aliphatic exhibit low toxicity toward fibroblast cells. Moreover, the viability and the proliferation of pancreatic β‐cells incubated inside hydrogels for 12 days are analyzed. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1548–1559     

Polyplex Particles Based on Comb‐Like Polyethylenimine/Poly(2‐ethyl‐2‐oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure

The present contribution is focused on feasibility of using comb‐like copolymers of polyethylenimine with poly(2‐ethyl‐2‐oxazoline) (LPEI‐comb‐PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well‐defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub‐100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.     

Acyl guanidine functional poly(2‐oxazoline)s as reactive intermediates and stimuli‐responsive materials

The many postpolymerization modification opportunities of biocompatible poly(2‐alkyl/aryl‐2‐oxazoline)s (PAOx), such as thiol–ene/thiol–yne, azide–alkyne cycloadditions, amidation, and transesterification, are one of the most appealing features of this polymer class for its popularity in biomedicine. Inspired by recent reports on guanidine‐catalyzed transesterification and amidation reactions of methyl ester substrates, we explored the use of guanidines as a reactant for the modification of methyl ester functional PAOx, to obtain the respective acyl guanidines. The obtained acyl guanidines functional polymers display reactivity toward α‐haloketones, yielding imidazole functional PAOx. The obtained polymer structures are protonated in a broad pH range, and the acyl guanidine moiety is demonstrated to be a cleavable linker under basic conditions. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019 , 57, 2616–2624     

Poly(2‐ethyl‐2‐oxazoline) as Alternative for the Stealth Polymer Poly(ethylene glycol): Comparison of in vitro Cytotoxicity and Hemocompatibility

Limitations of PEG in drug delivery have been reported from clinical trials. PEtOx (0.4–40 kDa) as alternative is synthesized by a living, microwave‐assisted polymerization, and is directly compared to PEG of similar molar mass regarding cytotoxicity and hemocompatibility. In short‐term treatments, both types of polymers are well tolerated even at high concentrations. Moderate concentration and molar mass dependent cytotoxic effects occurred only after long‐term incubation at concentrations higher than therapeutic doses. PEtOx possesses not only an easy route of synthesis and beneficial physicochemical characteristics such as low viscosity and high stability, which are advantageous over PEG, but additionally in vitro toxicology comparable to PEG.

     

Concentration effects in the cationic ring‐opening polymerization of 2‐ethyl‐2‐oxazoline in N,N‐dimethylacetamide

The monomer concentration for the cationic ring‐opening polymerization of 2‐ethyl‐2‐oxazoline in N,N‐dimethylacetamide was optimized utilizing high‐throughput experimentation methods. Detailed ¹H‐NMR spectroscopic investigations were performed to understand the mechanistic aspects of the observed concentration effects. Finally, the improved polymerization concentration was applied for the synthesis of higher molecular weight (> 10,000 Da) poly(2‐ethyl‐2‐oxazoline)s. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1487–1497, 2005     

Thermoresponsive polymeric nanoparticles based on poly(2‐oxazoline)s and tannic acid

Hydrogen bonding is widely present and plays a significant role in material science and supramolecular chemistry. This work reports a straightforward strategy for the preparation of polymeric nanoparticles from neutral poly(2‐oxazoline)s (POx) and tannic acid (TA) driven by their intermolecular hydrogen bonding. Dynamic light scattering (DLS) and scanning electron microscope (SEM) measurements showed that POx bearing different substituents, that is, methyl, ethyl and n‐propyl in the 2‐position all could assemble with TA into stable nanoparticles in water or ethanol. The diameter of the assembled nanoparticles could be manipulated by varying parameters such as molecular weight of POx, concentration and ratio of POx, and TA. Interestingly, POx/TA nanoparticles exhibited upper critical solution temperature (UCST)‐type thermoresponsive properties in ethanol or water depending on the molecular weight and substituent in the 2‐position of POx. Increasing or decreasing the temperature at the transition point resulted in the reversible transformation between assembled nanoparticles and disassembled poly(2‐n‐propyl‐2‐oxazoline) (PnPrOx) and TA. In view of the tailored size of the stable nanoparticles and the biocompatibilities of POx and TA, the prepared thermoresponsive nanoparticles are promising candidates as carriers for medicine toward related biomedical applications. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 1520–1527     

Characterization of different poly(2‐oxazoline) block copolymers by MALDI‐TOF MS/MS and ESI‐Q‐TOF MS/MS

A complete library of poly(2‐oxazoline) block copolymers was synthesized via cationic ring opening polymerization for the characterization by two different soft ionization techniques, namely matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF MS). In addition, a detailed characterization was performed by tandem MS to gain more structural information about the block copolymer composition and its fragmentation behavior. The fragmentation of the poly(2‐oxazoline) block copolymers revealed the desired polymer structure and possible side reactions, which could be explained by different mechanisms, like 1,4‐ethylene or hydrogen elimination and the McLafferty +1 rearrangement. Polymers with aryl side groups showed less fragmentation due to their higher stability compared to polymers with alkyl side groups. These insights represent a further step toward the construction of a library with fragments and their fragmentation pathways for synthetic polymers, following the successful examples in proteomics. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

Synthesis,characterization, and cross‐linking of a library of statistical copolymers based on 2‐“soy alkyl”‐2‐oxazoline and 2‐ethyl‐2‐oxazoline

The synthesis of statistical copolymers consisting of 2‐ethyl‐2‐oxazoline (EtOx) and 2‐“soy alkyl”‐2‐oxazoline (SoyOx) via a microwave‐assisted cationic ring‐opening polymerization procedure is described. The majority of the resulting copolymers revealed polydispersity indices below 1.30. The reactivity ratios (r_EtOx 1.4 ± 0.3; r_SoyOx = 1.7 ± 0.3) revealed a clustered monomer distribution throughout the polymer chains. The thermal and surface properties of the pEtOx‐stat‐SoyOx copolymers were analyzed before and after UV‐curing demonstrating the decreased chain mobility after cross‐linking. In addition, the cross‐linked materials showed shape‐persistent swelling upon absorption of water from the air, whereby as little as 5 mol % SoyOx was found to provide efficient cross‐linking. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5371,–5379, 2007     

Poly(cyclic imino ether)s Beyond 2‐Substituted‐2‐oxazolines

2‐Oxazolines (2‐OZO) are 5‐membered cyclic imino ethers whose cationic ring‐opening polymerization (CROP) mechanism and resulting polymer properties are extensively studied. However, also 6‐ and 7‐membered cyclic imino ethers can be polymerized via CROP. Together with the much less studied 4‐ and 5‐substituted main‐chain chiral poly(2‐oxazoline)s (P‐2‐OZO), these compounds are interesting monomers to enhance the versatility of (co)poly(cyclic imino ether)s. To emphasize the potential of such alternative cyclic imino ether monomers, we provide an overview on the polymerizations of 2‐oxazine (2‐OZI) and chiral 4‐ and 5‐substituted 2‐OZO as well as of selected properties of the resulting polymers. In addition, the hydrolysis of these polymers into the corresponding poly(alkylene imine)s will be addressed.

     

Solubility behavior of amphiphilic block and random copolymers based on 2‐ethyl‐2‐oxazoline and 2‐nonyl‐2‐oxazoline in binary water–ethanol mixtures

The solution properties of random and block copolymers based on 2‐ethyl‐2‐oxazoline (EtOx) and 2‐nonyl‐2‐oxazoline (NonOx) were investigated in binary solvent mixtures ranging from pure water to pure ethanol. The solubility phase diagrams for the random and block copolymers revealed solubility (after heating), insolubility, dispersions, micellization as well as lower critical solution temperature (LCST) and upper critical solution temperature behavior. The random and block copolymers containing over 60 mol % pNonOx were found to be solubilized in ethanol upon heating, whereas the dissolution temperature of the block copolymers was found to be much higher than for the random copolymers due to the higher extent of crystallinity. Furthermore, the block copolymer containing 10 mol % pNonOx exhibited a LCST in aqueous solution at 68.7 °C, whereas the LCST for the random copolymer was found to be only 20.8 °C based on the formation of hydrophobic microdomains in the block copolymer. The random copolymer displayed a small increase in LCST up to a solvent mixture of 9 wt % EtOH, whereas further increase of ethanol led to a decrease in LCST, which is probably due to the “water‐breaking” effect causing an increased attraction between ethanol and the hydrophobic part of the copolymer. In addition, the EtOx‐NonOx block copolymers revealed the formation of micelles and dynamic light scattering demonstrated that the micellar size is increasing with increasing the ethanol content due to the enhanced solubility of EtOx. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 515–522, 2009     

One‐pot synthesis of 2‐phenyl‐2‐oxazoline‐containing quasi‐diblock copoly(2‐oxazoline)s under microwave irradiation

The microwave‐assisted statistical copolymerization of 2‐phenyl‐2‐oxazoline with 2‐methyl‐2‐oxazoline or 2‐ethyl‐2‐oxazoline is discussed in this contribution. Kinetic studies of these statistical copolymerizations as well as reactivity ratio determinations were performed to investigate the monomer distribution in these copoly(2‐oxazoline)s, demonstrating the formation of quasi‐diblock copolymers. In addition, the synthesis of copolymer series with monomer concentrations ranging from 0 to 100 mol % is described. These copolymer series were characterized with ¹H NMR spectroscopy, gas chromatography, and gel permeation chromatography. Moreover, the glass‐transition temperatures and solubility of these copolymers were studied, and this revealing better mixing of poly(2‐methyl‐2‐oxazoline) (pMeOx) with poly(2‐phenyl‐2‐oxazoline) (pPhOx) than poly(2‐ethyl‐2‐oxazoline) (pEtOx) with poly(2‐phenyl‐2‐oxazoline) (pPhOx). © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 416–422, 2007.