A Perspective on the History and Current Opportunities of Aqueous RAFT Polymerization.

Reversible addition-fragmentation chain transfer (RAFT) polymerization has proven itself as a powerful polymerization technique affording facile control of molecular weight, molecular weight distribution, architecture, and chain end groups - while maintaining a high level of tolerance for solvent and monomer functional groups. RAFT is highly suited to water as a polymerization solvent, with aqueous RAFT now utilized for applications such as controlled synthesis of ultra-high molecular weight polymers, polymerization induced self-assembly, and biocompatible polymerizations, among others. Water as a solvent represents a non-toxic, cheap, and environmentally friendly alternative to organic solvents traditionally utilized for polymerizations. This, coupled with the benefits of RAFT polymerization, makes for a powerful combination in polymer science. This perspective provides a historical account of the initial developments of aqueous RAFT polymerization at the University of Southern Mississippi from the McCormick Research Group, details practical considerations for conducting aqueous RAFT polymerizations, and highlights some of the recent advances aqueous RAFT polymerization can provide. Finally, some of the future opportunities that this versatile polymerization technique in an aqueous environment can offer are discussed, and it is anticipated that the aqueous RAFT polymerization field will continue to realize these, and other exciting opportunities into the future.

Label-free characterization of organic nanocarriers reveals persistent single molecule cores for hydrocarbon sequestration.

Self-assembled molecular nanostructures embody an enormous potential for new technologies, therapeutics, and understanding of molecular biofunctions. Their structure and function are dependent on local environments, necessitating in-situ/operando investigations for the biggest leaps in discovery and design. However, the most advanced of such investigations involve laborious labeling methods that can disrupt behavior or are not fast enough to capture stimuli-responsive phenomena. We utilize X-rays resonant with molecular bonds to demonstrate an in-situ nanoprobe that eliminates the need for labels and enables data collection times within seconds. Our analytical spectral model quantifies the structure, molecular composition, and dynamics of a copolymer micelle drug delivery platform using resonant soft X-rays. We additionally apply this technique to a hydrocarbon sequestrating polysoap micelle and discover that the critical organic-capturing domain does not coalesce upon aggregation but retains distinct single-molecule cores. This characteristic promotes its efficiency of hydrocarbon sequestration for applications like oil spill remediation and drug delivery. Such a technique enables operando, chemically sensitive investigations of any aqueous molecular nanostructure, label-free.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests.

RNAi-based technologies are ideal for pest control as they can provide species specificity and spare nontarget organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[ N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal- Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) were found to be efficiently taken up by cells, and to drive highly efficient gene knockdown. These IPECs could also trigger target gene knockdown and moderate larval mortality when fed to S. frugiperda larvae. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid ß Aggregation.

GM1 ganglioside is known to promote amyloid-ß (Aß) peptide aggregation in Alzheimer's disease. The roles of the individual saccharides and their distribution in this process are not understood. Acrylamide-based glycomonomers with either ß-d-glucose or ß-d-galactose pendant groups were synthesized to mimic the stereochemistry of saccharides present in GM1 and characterized via 1H NMR and electrospray ionization mass spectrometry. Glycopolymers of different molecular weights were synthesized by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization and characterized by NMR and GPC. The polymers were used as models to investigate the effects of molecular weight and saccharide unit type on Aß aggregation via thioflavin-T fluorescence and PAGE. High molecular weight (∼350 DP) glucose-containing glycopolymers had a profound effect on Aß aggregation, promoting formation of soluble oligomers of Aß and limiting fibril production, while the other glycopolymers and negative control had little effect on the Aß propagation process.

Block ionomer complexes consisting of siRNA and aRAFT-synthesized hydrophilic-block-cationic copolymers II: The influence of cationic block charge density on gene suppression.

Block ionomer complex (BIC)-siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown.

Antimicrobial Peptide Mimicking Primary Amine and Guanidine Containing Methacrylamide Copolymers Prepared by Raft Polymerization.

Naturally occurring antimicrobial peptides (AMPs) display the ability to eliminate a wide variety of bacteria, without toxicity to the host eukaryotic cells. Synthetic polymers containing moieties mimicking lysine and arginine components found in AMPs have been reported to show effectiveness against specific bacteria, with the mechanism of activity purported to depend on the nature of the amino acid mimic. In an attempt to incorporate the antimicrobial activity of both amino acids into a single water-soluble copolymer, a series of copolymers containing lysine mimicking aminopropyl methacrylamide (APMA) and arginine mimicking guanadinopropyl methacrylamide (GPMA) were prepared via aqueous RAFT polymerization. Copolymers were prepared with varying ratios of the comonomers, with degree of polymerization of 35-40 and narrow molecular weight distribution to simulate naturally occurring AMPs. Antimicrobial activity was determined against Gram-negative and Gram-positive bacteria under conditions with varying salt concentration. Toxicity to mammalian cells was assessed by hemolysis of red blood cells and MTT assays of MCF-7 cells. Antimicrobial activity was observed for APMA homopolymer and copolymers with low concentrations of GPMA against all bacteria tested, with low toxicity toward mammalian cells.

Endolytic, pH-responsive HPMA-b-(L-Glu) copolymers synthesized via sequential aqueous RAFT and ring-opening polymerizations.

A facile synthetic pathway for preparing block copolymers with pH-responsive L-glutamic acid segments for membrane disruption is reported. Aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization was first used to prepare biocompatible, nonimmunogenic poly[N-(2-hydroxypropyl)methacrylamide]. This macro chain transfer agent (CTA) was then converted into a macroinitiator via simultaneous aminolysis and thiol-ene Michael addition using the primary amine substituted N-(3-aminopropyl)methacrylamide. This macroinitiator was subsequently utilized in the ring-opening polymerization of the N-carboxyanhydride monomer of γ-benzyl-L-glutamate. After deprotection, the pH-dependent coil-to-helix transformations of the resulting HPMA-b-(L-Glu) copolymers were monitored via circular dichroism spectroscopy. HPMA segments confer water solubility and biocompatibility while the L-glutamic acid repeats provide reversible coil-to-helix transitions at endosomal pH values (~5-6). The endolytic properties of these novel [HPMA-b-(L-Glu)] copolymers and their potential as modular components in drug carrier constructs was demonstrated utilizing red blood cell hemolysis and fluorescein release from POPC vesicles.

Antimicrobial poly(methacrylamide) derivatives prepared via aqueous RAFT polymerization exhibit biocidal efficiency dependent upon cation structure.

Antimicrobial peptides (AMPs) show great potential as alternative therapeutic agents to conventional antibiotics as they can selectively bind and eliminate pathogenic bacteria without harming eukaryotic cells. It is of interest to develop synthetic macromolecules that mimic AMPs behavior, but that can be produced more economically at commercial scale. Herein, we describe the use of aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare primary and tertiary amine-containing polymers with precise molecular weight control and narrow molecular weight distributions. Specifically, N-(3-aminopropyl)methacrylamide (APMA) was statistically copolymerized with N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) or N-[3-(diethylamino)propyl]methacrylamide (DEAPMA) to afford a range of (co)polymer compositions. Analysis of antimicrobial activity against E. coli (Gram-negative) and B. subtilis (Gram-positive) as a function of buffer type, salt concentration, pH, and time indicated that polymers containing large fractions of primary amine were most effective against both strains of bacteria. Under physiological pH and salt conditions, the polymer with the highest primary amine content caused complete inhibition of bacterial growth at low concentrations, while negligible hemolysis was observed over the full range of concentrations tested, indicating exceptional selectivity. The cytotoxicity of select polymers was evaluated against MCF-7 cells.

Guanidine-Containing Methacrylamide (Co)polymers via aRAFT: Toward a Cell Penetrating Peptide Mimic().

We report the synthesis and controlled radical homo- and block copolymerization of 3-guanidinopropyl methacrylamide (GPMA) utilizing aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization. The resulting homopolymer and block copolymer with N-(2-hydroxypropyl) methacrylamide (HPMA) were prepared to mimic the behavior of cell penetrating peptides (CPPs) and poly(arginine) (> 6 units) which have been shown to cross cell membranes. The homopolymerization mediated by 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl)pentanoic acid (CEP) in aqueous buffer exhibited pseudo-first-order kinetics and linear growth of molecular weight with conversion. Retention of the "living" thiocarbonylthio ω-end-group was demonstrated through successful chain extension of the GPMA macroCTA yielding GPMA(37)-b-GPMA(61) (M(w)/M(n) =1.05). Block copolymers of GPMA with the non-immunogenic, biocompatible HPMA were synthesized yielding HPMA(271)-b-GPMA(13) (M(w)/M(n) = 1.15). Notably, intracellular uptake was confirmed by fluorescence microscopy, confocal laser scanning microscopy, and flow cytometry experiments after 2.5 h incubation with KB cells at 4 °C and at 37 °C utilizing FITC-labeled, GPMA-containing copolymers. The observed facility of cellular uptake and the structural control afforded by aRAFT polymerization suggest significant potential for these synthetic (co)polymers as drug delivery vehicles in targeted therapies.

Tailored design of Au nanoparticle-siRNA carriers utilizing reversible addition-fragmentation chain transfer polymers.

The facile synthesis of polymer-stabilized Au nanoparticles (AuNPs) capable of forming neutral, sterically stable complexes with small interfering RNA (siRNA) is reported. The amine-containing cationic block of poly(N-2-hydroxypropyl methacrylamide(70)-block-N-[3-(dimethylamino)propyl] methacrylamide(24)) [P(HPMA(70)-b-DMAPMA(24))] was utilized to promote the in situ reduction of Au(3+) to AuNPs and subsequently bind small interfering RNA, while the nonimmunogenic, hydrophilic block provided steric stabilization. The ratio of [DMAPMA](0)/[Au(3+)](0) utilized in the reduction reaction was found to be critical to the production of polymer-stabilized AuNPs capable of complexing siRNA. Significant protection ( approximately 100 times) against nucleases was demonstrated by enzymatic tests, while gene down-regulation experiments indicated successful delivery of siRNA to cancerous cells.

Rational design of targeted cancer therapeutics through the multiconjugation of folate and cleavable siRNA to RAFT-synthesized (HPMA-s-APMA) copolymers.

A well-defined N-(2-hydroxypropyl)methacrylamide-s-N-(3-aminopropyl)methacrylamide (HPMA-s-APMA) copolymer, synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, was utilized for the rational design of multiconjugates containing both a gene therapeutic, small interfering RNA (siRNA), and a cancer cell targeting moiety, folate. The copolymer contains a biocompatible poly(HPMA) portion (91 mol %) and a primary amine, APMA, portion (9 mol %). A fraction (20 mol %) of the APMA repeats were converted to activated thiols utilizing the amine- and sulfhydryl-reactive molecule N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). 5'-Thiolated sense strand RNAs were then coupled to the polymer through a disulfide exchange with pendant pyridyldithio moieties, giving an 89 +/- 4% degree of conjugation. The unmodified APMA units (80 mol %) were subsequently coupled to amine reactive folates with 81 +/- 1% efficiency. This yielded a multiconjugate copolymer with 91 mol % HPMA, 2 mol % RNA, and 6 mol % folate. siRNA formation was achieved by annealing antisense strands to the conjugated RNA sense strands. Subsequent siRNA cleavage under intracellular conditions demonstrated the potential utility of this carrier in gene delivery. The multiconjugate copolymer and siRNA release were characterized by UV-vis spectroscopy and polyacrylamide gel electrophoresis.

Facile synthesis of multivalent folate-block copolymer conjugates via aqueous RAFT polymerization: targeted delivery of siRNA and subsequent gene suppression.

Cell specific delivery of small interfering ribonucleic acid (siRNA) using well-defined multivalent folate-conjugated block copolymers is reported. Primary amine functional, biocompatible, hydrophilic-block-cationic copolymers were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. N-(2-hydroxypropyl)methacrylamide) (HPMA), a permanently hydrophilic monomer, was copolymerized with a primary amine containing monomer, N-(3-aminopropyl)methacrylamide (APMA). Poly(HPMA) confers biocompatibility, while APMA provides amine functionality, allowing conjugation of folate derivatives. HPMA-stat-APMA was chain extended with a cationic block, poly(N-[3-(dimethylamino)propyl]methacrylamide), to promote electrostatic complexation between the copolymer and the negatively charged phosphate backbone of siRNA. Notably, poly(HPMA) stabilizes the neutral complexes in aqueous solution, while APMA allows the conjugation of a targeting moiety, thus, dually circumventing problems associated with the delivery of genes via cationically charged complexes (universal transfection). Fluorescence microscopy and gene down-regulation studies indicate that these neutral complexes can be specifically delivered to cancer cells that overexpress folate receptors.

Tailoring the network properties of Ca2+ crosslinked Aloe vera polysaccharide hydrogels for in situ release of therapeutic agents.

Properties of Aloe vera galacturonate hydrogels formed via Ca(2+) crosslinking have been studied in regard to key parameters influencing gel formation including molecular weight, ionic strength, and molar ratio of Ca(2+) to COO(-) functionality. Dynamic oscillatory rheology and pulsed field gradient NMR (PFG-NMR) studies have been conducted on hydrogels formed at specified Ca(2+) concentrations in the presence and absence of Na(+) and K(+) ions in order to assess the feasibility of in situ gelation for controlled delivery of therapeutics. Aqueous Ca(2+) concentrations similar to those present in nasal and subcutaneous fluids induce the formation of elastic Aloe vera polysaccharide (AvP) hydrogel networks. By altering the ratio of Ca(2+) to COO (-) functionality, networks may be tailored to provide elastic modulus (G') values between 20 and 20000 Pa. The Aloe vera polysaccharide exhibits time-dependent phase separation in the presence of monovalent electrolytes. Thus the relative rates of calcium induced gelation and phase separation become major considerations when designing a system for in situ delivery applications where both monovalent (Na(+), K(+)) and divalent (Ca(2+)) ions are present. PFG-NMR and fluorescence microscopy confirm that distinctly different morphologies are present in gels formed in the presence and absence of 0.15 M NaCl. Curve fitting of theoretical models to experimental release profiles of fluorescein labeled dextrans indicate diffusion rates are related to hydrogel morphology. These studies suggest that for efficient in situ release of therapeutic agents, polymer concentrations should be maintained above the critical entanglement concentration ( Ce, 0.60 wt %) when [Ca(2+)]/[COO(-)] ratios are less than 1. Additionally, the monovalent electrolyte concentration in AvP solutions should not exceed 0.10 M prior to Ca(2+) crosslinking.

Advances in the synthesis of amphiphilic block copolymers via RAFT polymerization: stimuli-responsive drug and gene delivery.

Controlled/'living' radical polymerization methods, including the versatile reversible addition-fragmentation chain transfer (RAFT) polymerization process, are rapidly moving to the forefront in construction of drug and gene delivery vehicles. The RAFT technique allows an unprecedented latitude in the synthesis of water soluble or amphiphilic architectures with precise dimensions and appropriate functionality for attachment and targeted delivery of diagnostic and therapeutic agents. This review focuses on the chemistry of the RAFT process and its potential for preparing well-defined block copolymers and conjugates capable of stimuli-responsive assembly and release of bioactive agents in the physiological environment. Recent examples of block copolymers with designed structures and segmental compositions responsive to changes in pH or temperature are reviewed and hurdles facing further development of these novel systems are discussed.

Thermoreversible hydrogels from RAFT-synthesized BAB triblock copolymers: steps toward biomimetic matrices for tissue regeneration.

Narrowly dispersed, temperature-responsive BAB block copolymers capable of forming physical gels under physiological conditions were synthesized via aqueous reversible addition fragmentation chain transfer (RAFT) polymerization. The use of a difunctional trithiocarbonate facilitates the two-step synthesis of BAB copolymers with symmetrical outer blocks. The outer B blocks of the triblock copolymers consist of poly(N-isopropylacrylamide) (PNIPAM) and the inner A block consists of poly(N,N-dimethylacrylamide). The copolymers form reversible physical gels above the phase transition temperature of PNIPAM at concentrations as low as 7.5 wt % copolymer. Mechanical properties similar to collagen, a naturally occurring polypeptide used as a three-dimensional in vitro cell growth scaffold, have been achieved. Herein, we report the mechanical properties of the gels as a function of solvent, polymer concentration, and inner block length. Structural information about the gels was obtained through pulsed field gradient NMR experiments and confocal microscopy.

Structural characterization and solution properties of a galacturonate polysaccharide derived from Aloe vera capable of in situ gelation.

A series of highly purified galacturonate polysaccharides have been extracted from the Aloe vera plant and analyzed in terms of chemical composition and molecular weight. This Aloe vera polysaccharide (AvP) has been found to exist as a high molecular weight species and possess a unique chemical composition, including a high galacturonic acid (GalA) content and low degree of methyl ester substitution. These factors facilitate gel formation upon exposure to low concentrations of calcium ions, leading to potential application in formulations designed for in situ nasal or subcutaneous protein delivery. Thorough examination of classic dilute solution properties, the [eta]-M(w), and R(g)-M(w) relationships, persistence length (L(p)), and inherent chain stiffness (B parameter), indicate an expanded random coil in aqueous salt solutions. The critical concentration for transition from dilute to concentrated solution, C(e), was determined by measuring both the zero shear viscosity (eta(o)) and fluorescence emission of the probe molecule 1,8-anilino-1-naphthalene sulfonic acid (1,8-ANS) as a function of polymer concentration. Examination of zeta potential and C(e) as a function of ionic strength indicates that the shift in C(e) from 0.60 to 0.30 wt % is related to an increased occurrence of intermolecular interactions at high salt concentrations. Additionally, dynamic rheology data are presented highlighting the ability of AvP to form gels at low polymer and calcium ion concentrations, exemplifying the technological potential of this polysaccharide for in situ drug delivery.

Facile synthetic procedure for omega, primary amine functionalization directly in water for subsequent fluorescent labeling and potential bioconjugation of RAFT-synthesized (co)polymers.

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.

Effect of sequential layer-by-layer surface modifications on the surface energy of plasma-modified poly(dimethylsiloxane).

Surface-initiated grafting of N,N-dimethylacrylamide, styrenesulfonate (SS), and (ar-vinylbenzyl)trimethylammonium chloride (VBTAC) from microwave plasma carboxylated, initiator-functionalized poly(dimethylsiloxane) (PDMS) surfaces was accomplished utilizing reversible addition-fragmentation chain transfer (RAFT) polymerization. Surface spectroscopic attenuated total reflectance (ATR) FT-IR analysis and atomic force microscopy (AFM) measurements were utilized to determine surface grafting and morphological surface features. The VBTAC-grafted PDMS provided a smooth, hydrophilic cationic surface for creating layer-by-layer (LBL) surfaces via alternating deposition of well-defined poly(SS) and poly(VBTAC), also prepared via aqueous RAFT. Comparisons of the ATR FT-IR spectra of the LBL assemblies and those of respective anionic poly(SS) and cationic poly(VBTAC) components confirmed strong electrostatic complexation of a fraction of the sulfonate and quarternary ammonium species in the layers as well as the existence of noncomplexed species. AFM images of surface topology indicated the presence of domains, likely phase-separated segments of the respective homopolymers, as well as interlayer mixing. The employed LBL methodology results in formation of stable, highly hydrophilic surfaces on a PDMS substrate. To our knowledge, this is the first study that illustrates surface functionalization of PDMS using microwave plasma and RAFT polymerization, followed by LBL deposition of polyelectrolytes.

Layer-by-layer assembly of pH-responsive, compositionally controlled (co)polyelectrolytes synthesized via RAFT.

Homo- and block copolyelectrolytes that have well-defined structures and are responsive to pH were synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization and employed to produce layer-by-layer (LBL) films. Acrylamido monomers with carboxylate, sulfonate, and amine functionality were utilized to provide both strong and weak homopolyelectrolytes and mixed strong/weak copolyelectrolyte systems. Multilayer films were prepared under specified conditions of pH and ionic strength and analyzed via atomic force microscopy and ellipsometry to study the effects of changes in the local molecular environment on film morphologies. The pH responsiveness and integrity of the multilayer assemblies were investigated by exposing films to solutions of varying pH in a fluid cell and performing in situ AFM analysis. The multilayer dimensions, morphology, and integrity were found to depend on the molecular architecture of the polyelectrolytes, with changes in segmental type and repeating unit distribution producing dramatic differences in film characteristics. These results suggest the possibility of producing LBL assemblies of precisely controlled dimensions and properties by specifically tailoring copolymer structure. To our knowledge, this is the first report of LBL assembly of RAFT-synthesized homo- and copolyelectrolyte multilayer complexes.