Linear Poly(ethylenimine-propylenimine) Random Copolymers for Gene Delivery: From Polymer Synthesis to Efficient Transfection with High Serum Tolerance.

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

Visualization and design of the functional group distribution during statistical copolymerization.

Even though functional copolymers with a low percentage of functional comonomer units (up to 20 mol%) are widely used, for instance for the development of polymer therapeutics and hydrogels, insights in the functional group distribution over the actual chains are lacking and the average composition is conventionally used to describe the functionalization degree. Here we report the visualization of the monomer distribution over the different polymer chains by a synergetic combination of experimental and theoretical analysis aiming at the construction of functionality-chain length distributions (FUNC-CLDs). A successful design of the chemical structure of the comonomer pair, the initial functional comonomer amount (13 mol%), and the temperature (100 °C) is performed to tune the FUNC-CLD of copoly(2-oxazoline)s toward high functionalization degree for both low (100) and high (400) target degrees of polymerization. The proposed research strategy is generic and extendable to a broad range of copolymerization chemistries, including reversible deactivation radical polymerization.

Unexpected Reactivity Switch in the Statistical Copolymerization of 2-Oxazolines and 2-Oxazines Enabling the One-Step Synthesis of Amphiphilic Gradient Copolymers.

Poly(2-oxazoline)s and, more recently, also poly(2-oxazine)s represent an emerging class of polymers with a broad range of applications. Surprisingly, to date, the statistical copolymerization of these two cyclic imino ether monomers has not yet been reported. Herein, we demonstrate that the statistical copolymerization of 2-oxazines with 2-oxazolines can lead to the formation of amphiphilic gradient copolymers in a single step. These gradient copolymers combine the high structural modularity of poly(2-oxazoline)s with the excellent biological properties of poly(2-oxazine)s, especially poly(2-methyl-2-oxazine). The copolymerization was found to proceed in a nonexpected way with the relative incorporation rates of the monomers being opposite to the reactivity observed for the corresponding homopolymerizations. In fact, the statistical copolymerizations lead to faster incorporation of the 2-oxazine followed by a gradual transition toward the 2-oxazoline. The self-assembly properties of the prepared amphiphilic poly[(2-methyl-2-oxazine)- grad-(2-butyl-2-oxazoline)] (PMeOzi- grad-PBuOx) as well as the thermoresponsive poly[(2-methyl-2-oxazine)- grad-(2-propyl-2-oxazoline)] (PMeOzi- grad-PPrOx) confirmed their potential as stimuli-responsive nonionic surfactants for various applications. Finally, the noncytotoxic character and cellular uptake of PMeOzi- grad-PBuOx copolymers was confirmed in vitro in SKOV3 cells.

Straightforward Route to Superhydrophilic Poly(2-oxazoline)s via Acylation of Well-Defined Polyethylenimine.

Herein, we describe a new method for the synthesis of superhydrophilic poly(2-alkyl-2-oxazoline)s (PAOx) from poly(2-ethyl-2-oxazoline) (PEtOx). A well-defined linear polyethylenimine was prepared from PEtOx by controlled acidic hydrolysis of its side-chains followed by reacylation with different carboxylic acids. Using this protocol, we obtained a series of new hydrophilic PAOx containing side-chain ether groups with potential in biomaterials science. The relative hydrophilicity of the polymers was assessed, revealing that poly(2-methoxymethyl-2-oxazoline) (PMeOMeOx) is the most hydrophilic PAOx reported to date. Additionally, the amorphous poly(2-methoxy-ethoxy-ethoxymethyl-2-oxazoline) (PDEGOx) shows the lowest reported glass transition temperature (-25 °C) within the PAOx family to date. The biomedical potential of the prepared polymers was further fortified by an in vitro cytotoxicity study, where all polymers appeared to be noncytotoxic. The described synthetic protocol is universal and can be extremely versatile, especially for PAOx that are difficult to prepare by conventional cationic ring-opening polymerization due to the monomer interference and/or degradation.

The Elusive Seven-Membered Cyclic Imino Ether Tetrahydrooxazepine.

Cyclic imino ether heterocycles are used as ligands in transition metal catalysis, in various drugs and as reactive monomers in living cationic ring-opening polymerization (CROP). While five- and six-membered cyclic imino ethers, i.e. 2-oxazolines and 4,5-dihydro-1,3-oxazines, have extensively been studied in these areas, their seven-membered ring counterparts have remained unexplored. Herein, we report the synthesis of 2-phenyl-4,5,6,7-tetrahydro-1,3-oxazepine allowing reassignment of the earlier, incorrectly reported 4,5,6,7-tetrahydro-1,3-oxazepines as their N-acylated pyrrolidine isomers. Finally, we also report a comparison of the CROP reactivity of a homologous series of cyclic imino ethers with a 2-carbon, 3-carbon, and 4-carbon methylene bridge, revealing a remarkable ring size effect.

Defined High Molar Mass Poly(2-Oxazoline)s.

Poly(2-alkyl-2-oxazoline)s (PAOx) are regaining interest for biomedical applications. However, their full potential is hampered by the inability to synthesise uniform high-molar mass PAOx. In this work, we proposed alternative intrinsic chain transfer mechanisms based on 2-oxazoline and oxazolinium chain-end tautomerisation and derived improved polymerization conditions to suppress chain transfer, allowing the synthesis of highly defined poly(2-ethyl-2-oxazoline)s up to ca. 50 kDa (dispersity (Ð) <1.05) and defined polymers up to at least 300 kDa (Ð<1.2). The determination of the chain transfer constants for the polymerisations hinted towards the tautomerisation of the oxazolinium chain end as most plausible cause for chain transfer. Finally, the method was applied for the preparation of up to 60 kDa molar mass copolymers of 2-ethyl-2-oxazoline and 2-methoxycarbonylethyl-2-oxazoline.

Chemical Design of Non-Ionic Polymer Brushes as Biointerfaces: Poly(2-oxazine)s Outperform Both Poly(2-oxazoline)s and PEG.

The era of poly(ethylene glycol) (PEG) brushes as a universal panacea for preventing non-specific protein adsorption and providing lubrication to surfaces is coming to an end. In the functionalization of medical devices and implants, in addition to preventing non-specific protein adsorption and cell adhesion, polymer-brush formulations are often required to generate highly lubricious films. Poly(2-alkyl-2-oxazoline) (PAOXA) brushes meet these requirements, and depending on their side-group composition, they can form films that match, and in some cases surpass, the bioinert and lubricious properties of PEG analogues. Poly(2-methyl-2-oxazine) (PMOZI) provides an additional enhancement of brush hydration and main-chain flexibility, leading to complete bioinertness and a further reduction in friction. These data redefine the combination of structural parameters necessary to design polymer-brush-based biointerfaces, identifying a novel, superior polymer formulation.

Tuning of Thermoresponsivity of a Poly(2-alkyl-2-oxazoline) Block Copolymer by Interaction with Surface-Active and Chaotropic Metallacarborane Anion.

Thermoresponsive nanoparticles based on the interaction of metallacarboranes, bulky chaotropic and surface-active anions, and poly(2-alkyl-2-oxazoline) block copolymers were prepared. Recently, the great potential of metallacarboranes have been recognized in biomedicine and many delivery nanosystems have been proposed. However, none of them are thermoresponsive. Therefore, a thermoresponsive block copolymer, poly(2-methyl-2-oxazoline)-block-poly(2-n-propyl-2-oxazoline) (PMeOx-PPrOx), was synthesized to encapsulate metallacarboranes. Light scattering, NMR spectroscopy, isothermal titration calorimetry, and cryogenic TEM were used to characterize all solutions of the formed nanoparticles. The cloud-point temperature (TCP ) of the block copolymer was observed at 30 °C and polymeric micelles formed above this temperature. Cobalt bis(dicarbollide) anion (COSAN) interacts with both polymeric segments. Depending on the COSAN concentration, this affinity influenced the phase transition of the thermoresponsive PPrOx block. The TCP shifted to lower values at a lower COSAN content. At higher COSAN concentrations, the hybrid nanoparticles are fragmented into relatively small pieces. This system is also thermoresponsive, whereby an increase in temperature leads to higher polymer mobility and COSAN release.

Fluorinated 2-Alkyl-2-oxazolines of High Reactivity: Spacer-Length-Induced Acceleration for Cationic Ring-Opening Polymerization As a Basis for Triphilic Block Copolymer Synthesis.

The synthesis of defined triphilic terpolymers with hydrophilic, lyophilic, and fluorophilic blocks is an important challenge as a basis for the development of multicompartment self-assembled structures with potential for, e.g., cascade catalysis and multidrug loading. The synthesis of fluorophilic poly(2-oxazoline)s generally suffers from a very low reactivity of fluorinated 2-oxazoline monomers in cationic ring-opening polymerization (CROP). We report a systematic study on overcoming the extremely low reactivity of 2-perfluoroalkyl-2-oxazolines in CROP by the insertion of methyl and ethyl hydrocarbon spacers between the 2-oxazoline ring and the trifluoromethyl group. The kinetic studies showed the gradual increase of the rate of polymerization with increasing of the hydrocarbon spacer length. The monomer with an ethyl spacer was found to have similar reactivity as 2-alkyl-2-oxazolines and allowed the synthesis of defined triphilic triblock copolymers.

Poly(2-ethyl-2-oxazoline) conjugates with doxorubicin for cancer therapy: In vitro and in vivo evaluation and direct comparison to poly[N-(2-hydroxypropyl)methacrylamide] analogues.

We designed and synthesized a new delivery system for the anticancer drug doxorubicin based on a biocompatible hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) carrier with linear architecture and narrow molar mass distribution. The drug is connected to the polymer backbone via an acid-sensitive hydrazone linker, which allows its triggered release in the tumor. The in vitro studies demonstrate successful cellular uptake of conjugates followed by release of the cytostatic cargo. In vivo experiments in EL4 lymphoma bearing mice revealed prolonged blood circulation, increased tumor accumulation and enhanced antitumor efficacy of the PEtOx conjugate having higher molecular weight (40 kDa) compared to the lower molecular weight (20 kDa) polymer. Finally, the in vitro and in vivo anti-cancer properties of the prepared PEtOx conjugates were critically compared with those of the analogous system based on the well-established PHPMA carrier. Despite the relatively slower intracellular uptake of PEtOx conjugates, resulting also in their lower cytotoxicity, there are no substantial differences in in vivo biodistribution and anti-cancer efficacy of both classes of polymer-Dox conjugates. Considering the synthetic advantages of poly(2-alkyl-2-oxazoline)s, the presented study demonstrates their potential as a versatile alternative to well-known PEO- or PHPMA-based materials for construction of drug delivery systems.

Block and Gradient Copoly(2-oxazoline) Micelles: Strikingly Different on the Inside.

Herein, we provide a direct proof for differences in the micellar structure of amphiphilic diblock and gradient copolymers, thereby unambiguously demonstrating the influence of monomer distribution along the polymer chains on the micellization behavior. The internal structure of amphiphilic block and gradient co poly(2-oxazolines) based on the hydrophilic poly(2-methyl-2-oxazoline) (PMeOx) and the hydrophobic poly(2-phenyl-2-oxazoline) (PPhOx) was studied in water and water-ethanol mixtures by small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), static and dynamic light scattering (SLS/DLS), and 1H NMR spectroscopy. Contrast matching SANS experiments revealed that block copolymers form micelles with a uniform density profile of the core. In contrast to popular assumption, the outer part of the core of the gradient copolymer micelles has a distinctly higher density than the middle of the core. We attribute the latter finding to back-folding of chains resulting from hydrophilic-hydrophobic interactions, leading to a new type of micelles that we refer to as micelles with a "bitterball-core" structure.

Cyclic Polymers: From Scientific Curiosity to Advanced Materials for Gene Delivery and Surface Modification.

Cyclic versus linear: The superiority of cyclic polymers over their linear counterparts is highlighted. Cyclic poly(2-oxazoline)s have been shown to provide excellent shielding properties when grafted to TiO2 surfaces and Fe3 O4 nanoparticles owing to their ultrahigh grafting densities leading to low friction surfaces, superior antifouling properties, and extreme nanoparticle stabilization.

Multireactive Poly(2-oxazoline) Nanofibers through Electrospinning with Crosslinking on the Fly.

Crosslinked hydrophilic poly(2-oxazoline)-based nanofibers amenable to facile multifunctionalization are fabricated using alkene-containing poly(2-alkyl-2-oxazoline)s (PAOx) via in situ photoinitiated radical thiol-ene crosslinking during electrospinning. The resulting crosslinked nanofibers are demonstrated to be multifunctionalizable using different chemistries as they contain two functional handles, being the alkene moieties from the parent copolymer and the residual thiol groups from the tetra-thiol-based crosslinker. While the thiol groups in these nanofibers could be passivated or conjugated to install functional molecules through thiol-maleimide conjugation, the alkene groups could sequentially be modified with thiol-containing molecules using photoinitiated radical thiol-ene reactions. Utilization of the photochemically induced conjugation of thiol-bearing molecules to the alkene groups on the nanofibers is used to obtain functionalization in a spatially controlled manner.

Continuous poly(2-oxazoline) triblock copolymer synthesis in a microfluidic reactor cascade.

Cationic ring-opening polymerizations of 2-oxazolines were investigated in continuous microflow reactors. Fast homopolymerizations of 2-ethyl-2-oxazoline (EtOx) and 2-n-propyl-2-oxazoline (nPropOx) were carried out up to 180 °C, yielding well-controlled polymers. Also well-defined diblock and triblock copolymers were produced in a microfluidic reactor cascade, demonstrating the high value of microflow synthesis for the built-up of advanced poly(2-oxazoline)-based polymers.

Sulfolane as Common Rate Accelerating Solvent for the Cationic Ring-Opening Polymerization of 2-Oxazolines.

The search for alternative solvents for the cationic ring-opening polymerization (CROP) of 2-methyl-2-oxazoline (MeOx) is driven by the poor solubility of P(MeOx) in polymerization solvents such as acetonitrile (CH3CN) and chlorobenzene as well as in MeOx itself. In this study, solvent screening has revealed that especially sulfolane is a good solvent for PMeOx. Unexpectedly, an increased propagation rate constant (kp) was found for the CROP of MeOx in sulfolane. Further extended kinetic studies at different temperatures (60-180 °C), revealed that the acceleration is due to an increase in frequency factor, while the activation energy (Ea) of the reaction is hardly affected. In order to explore the versatility of sulfolane as polymerization solvent for the CROP of 2-oxazolines in general, also the polymerization kinetics of other 2-oxazoline monomers, such as 2-ethyl-2-oxazoline (EtOx) and 2-phenyl-2-oxazoline (PhOx), have been studied, revealing a common acceleration of the CROP of 2-oxazoline monomers in sulfolane. This also enabled more controlled synthesis of PMeOx-block-PPhOx block copolymers that otherwise suffers from solvent incompatibility.

Total synthesis of (+/-)-frondosin B and (+/-)-5-epi-liphagal by using a concise (4+3) cycloaddition approach.

A recently developed (4+3) cycloaddition between dienes and furfuryl alcohols, as precursors of oxyallyl-type cations, has been used as a key step in the racemic syntheses of two natural products: frondosin B and liphagal. This work demonstrates the synthetic potential of this cycloaddition reaction, and offers a short synthetic route to an interesting family of natural products. A full account of these synthetic studies is presented, further illustrating the mechanism, scope, and limitations of this straightforward synthetic method for seven-membered rings.