Comparison between the LCST and UCST Transitions of Double Thermoresponsive Diblock Copolymers: Insights into the Behavior of POEGMA in Alcohols

Doubly thermoresponsive polymers consisting of a poly­[oligo­(ethylene glycol) methyl ether methacrylate] (POEGMA) block displaying UCST behavior in alcohols and a block of poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) or poly­(<i>N</i>,<i>N</i>-diethylacrylamide) (PDEAM), each of which has an LCST in water, were synthesized using RAFT polymerization followed by simultaneous activated ester/amine and nucleophilic thiol–ene postpolymerization conversions. Upon heating aqueous solutions of POEGMA-<i>b</i>-PNIPAM, ¹H NMR spectroscopy confirmed a sudden decrease of the PNIPAM signals at the LCST, indicating dehydration and chain collapse. Dynamic light scattering (DLS) and turbidity measurements observed the macroscopic phase separation of the PNIPAM block at the same temperature. In 2-propanol, ¹H NMR spectroscopy showed a gradual decrease of the POEGMA signals over a range of more than 30 °C during its UCST transition, indicating early stages of chain crumpling up to 20 °C above the macroscopic phase separation. The OEG side chains were found to collapse onto the backbone starting at the ester linkages, indicating the most unfavorable enthalpic polymer–solvent interactions occur adjacent to the ester group. Although the diblock copolymers displayed a strong concentration-dependent cloud point, ¹H NMR spectroscopy revealed a concentration-independent desolvation, indicating the potential for applications that are not based on phase separation but on changes of polymer conformation. The phase separation occurred within a narrow temperature range of ∼6 °C as evidenced by turbidity and DLS. This transition could be exploited to self-assemble POEGMA-<i>b</i>-PDEAM into micellar structures with POEGMA cores in 1-octanol. Cooling to ∼15 °C below the cloud point was necessary to produce compact structures. Upon heating, the aggregates remained compact until redissolving entirely within a range of 1 °C, making the UCST of POEGMA in alcohols a valuable tool for reversible self-assembly applications

Nitric Oxide (NO) Endows Arylamine-Containing Block Copolymers with Unique Photoresponsive and Switchable LCST Properties

The fabrication of materials that are responsive to endogenous gasotransmitter molecules (i.e., nitric oxide, hydrogen sulfide, and carbon monoxide) has emerged as an area of increasing research interest. In the case of nitric oxide (NO), <i>o</i>-phenylene­diamine derivatives have traditionally been employed due to their ability to react with NO in the presence of oxygen (O₂) with the formation of benzotriazole residues. Herein, we report the synthesis of a novel NO-responsive polymer containing aromatic primary amine groups derived from <i>p</i>-phenylene­diamine groups (i.e., isomers of <i>o</i>-phenylene­diamine). A new NO-responsive monomer, <i>N</i>-(4-amino­phenyl)­methacrylamide (<i>p</i>-NAPMA), was first synthesized via the amidation of one of the primary amine groups in the <i>p</i>-phenylene­diamine with methacrylic anhydride. Notably, the <i>p</i>-NAPMA monomer can efficiently react with NO in aqueous solution in the presence of O₂ with the generation of phenyl­diazonium groups rather than benzotriazole moieties. While the resultant phenyl­diazonium residues were relatively stable in aqueous solution, they were highly sensitive to UV irradiation (i.e., λ_max = 365 nm) which gave the formation of phenol derivatives. After incorporation into a thermoresponsive block copolymer using reversible addition–fragmentation chain transfer (RAFT) polymerization, the resulting diblock copolymer, poly­(ethylene glycol)-<i>b</i>-(<i>N</i>-isopropyl­acrylamide-<i>co</i>-<i>p</i>-NAPMA) (PEG-<i>b</i>-P­(NIPAM-<i>co</i>-<i>p</i>-NAPMA)), was rendered with unique NO- and UV-responsive characteristics. Specifically, the NO-triggered transformation of <i>p</i>-NAPMA moieties into phenyl­diazonium residues dramatically elevated the lower critical solution temperature (LCST) of the block copolymer due to increased water solubility of phenyl­diazonium residues at neutral pH (i.e., pH 7.4). Further, subsequent UV irradiation significantly decreased the LCST due to the formation of relatively hydrophobic phenol derivatives from the hydrophilic phenyl­diazonium intermediate. These results demonstrate, for the first time, that NO-responsive polymers can be synthesized without the necessity of incorporating <i>o</i>-phenylene­diamine groups and that a further solubility switch can be stimulated by irradiation with ultraviolet light

Application of Heterocyclic Polymers in the Ratiometric Spectrophotometric Determination of Fluoride

Herein we report the use of heterocyclic functional polymers in the ratiometric spectrophotometric determination of fluoride (F^–). Polymers incorporating benzo­[d]­[1,2,3]­triazole moieties linked to the polymer backbone via urea links are demonstrated to have utility for the ratiometric detection of the F^– ion, with a detection limit in the order of ∼2 μM. The hydrogen-bonding recognition between the benzo­[d]­[1,2,3]­triazole moiety and F^– ion was investigated using UV–vis spectrophotometry and NMR analysis. The importance of the urea linkage was elucidated by investigating a second benzo­[d]­[1,2,3]­triazole functional monomer wherein the heterocyclic group is attached to the polymerizable group via a carbamate linkage. The replacement of the urea link with a carbamate group led to significantly reduced F^– sensitivity. Moreover, by examining an analogous benzo­[d]­imidazole monomer it was demonstrated that having a nitrogen atom in the 2-position of the heterocycle was important for maximizing the sensitivity of the assay. Taken together, these results demonstrated that the urea-substituted benzo­[d]­[1,2,3]­triazole motif greatly enhances F^– ion detection. Importantly, the F^– ion sensing capability of the monomer is retained after incorporating into a diblock copolymer using reversible addition–fragmentation chain transfer (RAFT) polymerization

Assessment of Cholesterol-Derived <i>Ionic</i> Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived <i>ionic</i> copolymers were previously synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization as ‘smart’ gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly­(methacrylic acid-co-cholesteryl methacrylate) (P­(MAA-<i>co</i>-CMA)) and cationic poly­(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P­(DMAEMA-<i>co</i>-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P­(DMAEMA-<i>co</i>-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P­(MAA-<i>co</i>-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P­(MAA-<i>co</i>-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P­(MAA-<i>co</i>-CMA)-DNA) and 20:1 (Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P­(MAA-<i>co</i>-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P­(DMAEMA-<i>co</i>-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA particles are extremely promising candidates for <i>in vivo</i> applications of siRNA therapy

In Situ Formation of Polymer–Gold Composite Nanoparticles with Tunable Morphologies

A simple and efficient route to gold–polymer nanoparticle composites is described. Our versatile synthetic route exerts facile control over polymer nanoparticle morphology, including micelles, rod-like structures, and vesicles, all easily attainable from a single polymerization taken to different monomer conversions. Specifically, poly­[oligo­(ethylene glycol) methacrylate]-<i>b</i>-poly­(dimethylaminoethyl methacrylate)-<i>b</i>-poly­(styrene) (POEGMA-<i>b</i>-PDMAEMA-<i>b</i>-PST) triblock copolymers were synthesized using a polymerization induced self-assembly (PISA) approach. Subsequently, spherical gold nanoparticles (10 nm AuNPs) were formed at the hydrophilic–hydrophobic nexus of the assembled triblock copolymer nanoaggregates by the addition of chloroauric acid (HAuCl₄) followed by in situ reduction using NaBH₄. After reduction, the cloudy white nanoparticle dispersions turned to a red-purple color. The gold nanoparticles that formed were stabilized by the enveloping polymeric nanostructures, neither precipitation nor agglomeration occurred. We demonstrated that we were able to tune the gold nanoparticle composition in these polymer–gold composites by varying the concentration of chloroauric acid. Morphology, particle size, molecular weight, AuNP content, and chemical structure of the polymer structures were characterized by transmittance electron microscopy (TEM), dynamic light scattering (DLS), size exclusion chromatography (SEC), thermal gravimetric analysis (TGA), and ¹H NMR. Finally, the formation of the AuNPs occurred without affecting the polymer nanoparticle morphology

Polymerization-Induced Self-Assembly: The Effect of End Group and Initiator Concentration on Morphology of Nanoparticles Prepared via RAFT Aqueous Emulsion Polymerization

Polymerization-induced self-assembly (PISA) is a widely used technique for the synthesis of nanoparticles with various morphologies including spheres, worms, and vesicles. The development of a PISA formulation based on reversible addition–fragmentation chain transfer (RAFT) aqueous emulsion polymerization offers considerable advantages such as enhanced rate of polymerization, high conversion and environmentally friendly conditions. However, this formulation has typically produced spheres as opposed to worms and vesicles. Herein, we report the formation of vesicle morphology by increasing the RAFT end-group hydrophobicity of the macromolecular chain transfer agent or manipulating the radical initiator concentration used in the aqueous emulsion polymerization PISA formulation. Additionally, decreasing the molecular weight of the hydrophobic polystyrene domain in these vesicles leads to the formation of worms. This work demonstrates that RAFT end-group hydrophobicity and radical initiator concentration are key parameters which can be exploited to enable access to sphere, worm, and vesicle morphologies via the RAFT aqueous emulsion polymerization

RAFT Synthesis and Aqueous Solution Behavior of Novel pH- and Thermo-Responsive (Co)Polymers Derived from Reactive Poly(2-vinyl-4,4-dimethylazlactone) Scaffolds

Well-defined homopolymers of 2-vinyl-4,4-dimethylazlactone (VDA) and AB diblock copolymers of VDA with <i><i>N</i></i>,<i><i>N</i></i>-dimethylacrylamide (DMA) and <i>N</i>-isopropyl­acrylamide (NIPAM) prepared by reversible addition–fragmentation chain transfer (RAFT) radical polymerization are reported. VDA homopolymers reacted with <i><i>N</i></i>,<i><i>N</i></i>-dimethylethylenediamine (DMEDA), <i><i>N</i></i>,<i><i>N</i>-</i>diethylethylenediamine (DEEDA), and picoylamine (PA) give novel tertiary amine functional polymers that exhibit inverse temperature aqueous solution characteristics in the case of the DMEDA and DEEDA derivatives (provided they are not protonated) and a pH-dependent solubility for the PA speciesit is soluble at low solution pH but becomes hydrophobic at ca. pH 4.0. VDA-DMA/NIPAM AB diblock copolymers are also readily modified with DMEDA, DEEDA, and PA to give a novel series of stimulus responsive block copolymers including tunably amphiphilic and schizophrenic species. DMEDA-DMA and DEEDA-DMA/NIPAM block copolymer derivatives undergo reversible temperature induced self-assembly in aqueous media by virtue of the inverse temperature solubility characteristics associated with these tertiary amino species. The aggregation behavior of these species is characterized using a combination of dynamic light scattering (DLS), ¹H NMR spectroscopy and transmission electron microscopy (TEM). For the PA derivatives, schizophrenic behavior is demonstrated in AB block copolymers with NIPAM with normal and inverse micelles being readily accessible simply by controlling the solution pH or temperature. Self-assembled species derived from a DMEDA-DMA block copolymer, containing tertiary amino functionality in the core, can be readily core cross-linked, locking the self-assembled structure, using 1,10-dibromodecane as evidenced by DLS. The ability of examples of the ‘smart’ block copolymers to sequester hydrophobic Nile Red upon application of a pH or temperature stimulus from an aqueous environment is also demonstrated. Finally, we show how, if desired, the DMEDA homopolymers can be further modified via the facile reaction with 1,3-propanesultone yielding the sulfopropylbetaine analogous materials

Effectively Delivering a Unique Hsp90 Inhibitor Using Star Polymers

We report the synthesis of a novel heat shock protein 90 (hsp90) inhibitor conjugated to a star polymer. Using reversible addition–fragmentation chain-transfer (RAFT) polymerization, we prepared star polymers comprising PEG attached to a predesigned functional core. The stars were cross-linked using disulfide linkers, and a tagged version of our hsp90 inhibitor was conjugated to the polymer core to generate nanoparticles (14 nm). Dynamic light scattering showed that the nanoparticles were stable in cell growth media for 5 days, and high-performance liquid chromatography (HPLC) analysis of compound-release at 3 different pH values showed that release was pH dependent. Cell cytotoxicity studies and confocal microscopy verify that our hsp90 inhibitor was delivered to cells using this nanoparticle delivery system. Further, delivery of our hsp90 inhibitor using star polymer induces apoptosis by a caspase 3-dependent pathway. These studies show that we can deliver our hsp90 inhibitor effectively using star polymers and induce apoptosis by the same pathway as the parent compound

Transformation of RAFT Polymer End Groups into Nitric Oxide Donor Moieties: En Route to Biochemically Active Nanostructures

Polymers with a terminal <i>S</i>-nitrosothiol moiety were synthesized by modifying the thiocarbonylthio end group formed by reversible addition–fragmentation chain transfer polymerization. Specifically, benzodithioate-terminated poly­[oligo­(ethylene glycol) methyl ether methacrylate] (POEGMA) was first synthesized by polymerizing OEGMA in the presence of 4-cyano-4-(phenylcarbonothioylthio)­pentanoic acid. Sequential treatment with hydrazine hydrate and a stoichiometric amount of nitrous acid resulted in the formation of <i>S</i>-nitrosothiol-terminated polymers. A similar approach was applied to block copolymers of POEGMA incorporating a domain of poly­[(<i>N</i>,<i>N</i>-diisopropylamino)­ethyl methacrylate], thus, enabling the preparation of pH responsive nitric oxide (NO)-releasing micelles. The micelles possessed substantially modified <i>S</i>-nitrosothiol loss kinetics compared to the hydrophilic homopolymer analogue. Moreover, thiol-triggered degradation of the <i>S</i>-nitrosothiol was significantly slower when the <i>S</i>-nitrosothiol was embedded in a micellar structure. These results demonstrate that it is possible to incorporate nitric oxide donor moieties directly onto a polymer chain end, enabling simple synthesis of biochemically active nanostructures

An Efficient and Highly Versatile Synthetic Route to Prepare Iron Oxide Nanoparticles/Nanocomposites with Tunable Morphologies

We report a versatile synthetic method for the <i>in situ</i> self-assembly of magnetic-nanoparticle-functionalized polymeric nanomorphologies, including spherical micelles and rod-like and worm-like micelles and vesicles. Poly­(oligoethylene glycol methacrylate)-<i>block</i>-(methacrylic acid)-<i>block</i>-poly­(styrene) (POEGMA-<i>b</i>-PMAA-<i>b</i>-PST) triblock copolymer chains were simultaneously propagated and self-assembled via a polymerization-induced self-assembly (PISA) approach. Subsequently, the carboxylic acid groups in the copolymers were used to complex an iron ion (Fe^II/Fe^III) mixture. Iron oxide nanoparticles were then formed in the central block, within the polymeric nanoparticles, via alkaline coprecipitation of the iron­(II) and (III) salts. Nanoparticle morphologies, particle sizes, molecular weights, and chemical structures were then characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), size exclusion chromatography (SEC), and ¹H NMR measurements. TEM micrographs showed that the average size of the magnetic nanoparticles was ∼7 nm at the hydrophobic/hydrophilic nexus contained within the nanoparticles. In addition, XRD was used to confirm the formation of iron oxide nanoparticles. Importantly, the polymeric nanoparticle morphologies were not affected by the coprecipitation of the magnetic nanoparticles. The hybrid nanoparticles were then evaluated as negative MRI contrast agents, displaying remarkably high transverse relaxivities (<i>r</i>₂, greater than 550 mM^–1 s^–1 at 9.4 T); a result, that we hypothesize, ensues from iron oxide nanoparticle clustering at the hydrophobic–hydrophilic interface. This simple synthetic procedure is highly versatile and produces nanocarriers of tunable size and shape with high efficacy as MRI contrast agents and potential utility as theranostic delivery vectors