Selective and Rapid Light-Induced RAFT Single Unit Monomer Insertion in Aqueous Solution.

The photocatalyst Zn(II) meso-tetra(4-sulfonatophenyl)porphyrin (ZnTPPS) is found to substantially accelerate visible-light-initiated (red, yellow, green light) single unit monomer insertion (SUMI) of N,N-dimethylacrylamide into the reversible addition-fragmentation chain transfer (RAFT) agent, 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (RAFT1 ), in aqueous solution. Thus, under irradiation with red (633 nm) or yellow (593 nm) light with 50 mpm (moles per million mole of monomer) ZnTPPS at 30 °C, the rate enhancement provided by photoinduced energy or electron transfer (PET) is ≈sevenfold over the rate of direct photoRAFT-SUMI (without catalyst), which corresponds to achieving full and selective reaction in hours versus days. Importantly, the selectivity, as judged by the absence of oligomers, is retained. Under green light at similar power, higher rates of SUMI are also observed. However, the degree of enhancement provided by PET-RAFT-SUMI over direct photoRAFT-SUMI as a function of catalyst concentration is less and some oligomers are formed.

Engineering the Biointerface of Electrospun 3D Scaffolds with Functionalized Polymer Brushes for Enhanced Cell Binding.

Electrospun ultrafine fibers prepared using a blend of poly(lactide- co-glycolide) (PLGA) and bromine terminated poly(l-lactide) (PLA-Br), were surface modified using surface-initiated (SI) Cu(0) mediated polymerization. Copolymers based on N-acryloxysuccinimide (NAS) and a low fouling monomer (either N, N-dimethylacrylamide (DMA), N-(2-hydroxypropyl)acrylamide (HPA), or N-acryloylmorpholine (NAM)) were grafted from the fiber surface to impart surface functionality and to reduce nonspecific protein adsorption. Inclusion of the functional NAS monomer facilitated the conjugation of a nonbioactive cyclic RAD peptide and a bioactive cyclic RGD peptide, the latter expected to facilitate cell adhesion through its affinity for the αvß3 integrin receptor. A detailed analysis of the surface of the electrospun fiber scaffolds in nongrafted form compared to the surface functionalized state is presented. Characteristic amino acid peaks are observed for both conjugated RGD and RAD peptides. Cell culture experiments confirmed cell specific attachment mediated through the presence of the bioactive RGD peptide mainly at high surface density.

Triple Activity of Lamivudine Releasing Sulfonated Polymers against HIV-1.

In this article a library of polymeric therapeutic agents against the human immunodeficiency virus (HIV) is presented. The library of statistical copolymers of varied molar mass was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The synthesized polymers comprise pendent hydroxyl and sulfonated side chains as well as the reverse transcriptase prodrug lamivudine (3TC) attached via a disulfide self-immolative linker. The glutathione mediated release of 3TC is demonstrated as well as the antiviral efficacy against HIV entry and polymerase activity. Although a high degree of polymer sulfonation is required for effective HIV entry inhibition, polymers with approximately ∼50% sulfonated monomer demonstrated potent kinase independent reverse transcriptase inhibition. In addition, the sulfonated polymers demonstrate activity against DNA-DNA polymerase, which suggests that these polymers may exhibit activity against a broad spectrum of viruses. In summary, the polymers described provide a triple-active arsenal against HIV with extracellular activity via entry inhibition and intracellular activity by kinase-dependent lamivudine-based and kinase-independent sulfonated polymer based inhibition. Since these sulfonated copolymers are easily formulated into gels, we envision them to be particularly suited for topical application to prevent the mucosal transmission of viruses, particularly HIV.

Novel RAFT amphiphilic brush copolymer steric stabilisers for cubosomes: poly(octadecyl acrylate)-block-poly(polyethylene glycol methyl ether acrylate).

Copolymers, particularly Pluronics®, are typically used to sterically stabilise colloidal nanostructured particles composed of a lyotropic liquid crystalline bicontinuous cubic phase (cubosomes). There is a need to design and assess new functionalisable stabilisers for these colloidal drug delivery systems. Six amphiphilic brush copolymers, poly(octadecyl acrylate)-block-poly(polyethylene glycol methyl ether acrylate) (P(ODA)-b-P(PEGA-OMe)), synthesised by reversible addition-fragmentation chain transfer (RAFT), were assessed as novel steric stabilisers for cubosomes. It was found that increasing the density of PEG on the nanostructured particle surface by incorporating a PEG brush design (i.e., brush copolymer), provided comparable and/or increased stabilisation effectiveness compared to a linear PEG structure, Pluronic® F127, which is extensively used for steric stabilisation of cubosomes. Assessment was conducted both prior to and following the removal of the dodecyl trithiocarbonate end-group, by free radical-induced reduction. The reduced (P(ODA)-b-P(PEGA-OMe) copolymers were more effective steric stabilisers for phytantriol and monoolein colloidal particle dispersions than their non-reduced analogues. High throughput characterisation methodologies, including an accelerated stability assay (ASA) and synchrotron small angle X-ray scattering (SAXS), were implemented in this study for the rapid assessment of steric stabiliser effectiveness and lyotropic liquid crystalline phase identification. Phytantriol cubosomes stabilised with P(ODA)-b-P(PEGA-OMe) copolymers exhibited a double diamond cubic phase (Q(2)(D)), whilst monoolein cubosomes exhibited a primitive cubic phase (Q(2)(P)), analogous to those formed using Pluronic® F127.

Layer-by-layer polymer coating on discrete particles of cubic lyotropic liquid crystalline dispersions (cubosomes).

Cubic phase lyotropic liquid crystalline colloidal dispersions (cubosomes) were surface-modified with seven polyelectrolyte layers using a layer-by-layer (LbL) approach. The first layer consisted of a copolymer synthesized from methacrylic acid and oleoyl methacrylate for enhanced incorporation within the bilayer of the cubic nanostructure. Six additional layers of poly(L-lysine) and poly(methacrylic acid) were then sequentially added, followed by a washing procedure to remove polymer aggregates from the soft matter particles. Polymer buildup was monitored via microelectrophoresis, dynamic light scattering, and small-angle X-ray scattering. Polymer-coated cubosomes were observed with cryo-transmission electron microscopy. A potential application of the modified nanostructured particles presented in this study is to reduce the burst-release effect associated with drug-loaded cubosomes. The effectiveness of this approach was demonstrated through loading and release results from a model hydrophilic small molecule (fluorescein).

Immobilization and intracellular delivery of an anticancer drug using mussel-inspired polydopamine capsules.

We report a facile approach to immobilize pH-cleavable polymer-drug conjugates in mussel-inspired polydopamine (PDA) capsules for intracellular drug delivery. Our design takes advantage of the facile PDA coating to form capsules, the chemical reactivity of PDA films, and the acid-labile groups in polymer side chains for sustained pH-induced drug release. The anticancer drug doxorubicin (Dox) was conjugated to thiolated poly(methacrylic acid) (PMA(SH)) with a pH-cleavable hydrazone bond, and then immobilized in PDA capsules via robust thiol-catechol reactions between the polymer-drug conjugate and capsule walls. The loaded Dox showed limited release at physiological pH but significant release (over 85%) at endosomal/lysosomal pH. Cell viability assays showed that Dox-loaded PDA capsules enhanced the efficacy of eradicating HeLa cancer cells compared with free drug under the same assay conditions. The reported method provides a new platform for the application of stimuli-responsive PDA capsules as drug delivery systems.

Photoinitiated alkyne-azide click and radical cross-linking reactions for the patterning of PEG hydrogels.

The photolithographical patterning of hydrogels based solely on the surface immobilization and cross-linking of alkyne-functionalized poly(ethylene glycol) (PEG-tetraalkyne) is described. Photogenerated radicals as well as UV absorption by a copper chelating ligand result in the photochemical redox reduction of Cu(II) to Cu(I). This catalyzes the alkyne-azide click reaction to graft the hydrogels onto an azide-functionalized plasma polymer (N(3)PP) film. The photogenerated radicals were also able to abstract hydrogen atoms from PEG-tetraalkyne to form poly(α-alkoxy) radicals. These radicals can initiate cross-linking by addition to the alkynes and intermolecular recombination to form the PEG hydrogels. Spatially controlling the two photoinitiated reactions by UV exposure through a photomask leads to surface patterned hydrogels, with thicknesses that were tunable from tens to several hundreds of nanometers. The patterned PEG hydrogels (ca. 60 µm wide lines) were capable of resisting the attachment of L929 mouse fibroblast cells, resulting in surfaces with spatially controlled cell attachment. The patterned hydrogel surface also demonstrated spatially resolved chemical functionality, as postsynthetic modification of the hydrogels was successfully carried out with azide-functionalized fluorescent dyes via subsequent alkyne-azide click reactions.

Poly(vinyl alcohol) physical hydrogels: noncryogenic stabilization allows nano- and microscale materials design.

Physical hydrogels based on poly(vinyl alcohol), PVA, have an excellent safety profile and a successful history of biomedical applications. However, highly inhomogeneous and macroporous internal organization of these hydrogels as well as scant opportunities in bioconjugation with PVA have largely ruled out micro- and nanoscale control and precision in materials design and their use in (nano)biomedicine. To address these shortcomings, herein we report on the assembly of PVA physical hydrogels via "salting-out", a noncryogenic method. To facilitate sample visualization and analysis, we employ surface-adhered structured hydrogels created via microtransfer molding. The developed approach allows us to assemble physical hydrogels with dimensions across the length scales, from ∼100 nm to hundreds of micrometers and centimeter sized structures. We determine the effect of the PVA molecular weight, concentration, and "salting out" times on the hydrogel properties, i.e., stability in PBS, swelling, and Young's modulus using exemplary microstructures. We further report on RAFT-synthesized PVA and the functionalization of polymer terminal groups with RITC, a model fluorescent low molecular weight cargo. This conjugated PVA-RITC was then loaded into the PVA hydrogels and the cargo concentration was successfully varied across at least 3 orders of magnitude. The reported design of PVA physical hydrogels delivers methods of production of functionalized hydrogel materials toward diverse applications, specifically surface mediated drug delivery.

Surface "click" chemistry on brominated plasma polymer thin films.

A brominated plasma polymer (BrPP) thin film was fabricated on a variety of substrate surfaces (silicon wafers, glass, gold, and polymers) via the radio frequency glow discharge of 1-bromopropane. This BrPP thin film was highly adherent and stable and was found to be a useful platform for secondary reactions, leading to surfaces with specific chemical functionalities. Following nucleophilic exchange, an azide-functionalized PP thin film was prepared that was reactive toward two different alkynes via the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a paradigm of "click" chemistry. "Click" microcontact printing (microCP) of a fluorescent alkyne was also successfully carried out, demonstrating the versatility and functionality of this new class of reactive thin film plasma polymer coatings.

Noncovalent liposome linkage and miniaturization of capsosomes for drug delivery.

We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ∼1 µm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to ∼2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.

Bacterial membrane permeability of antimicrobial polymethacrylates: Evidence for a complex mechanism from super-resolution fluorescence imaging.

Amphiphilic polymers bearing cationic moieties are an emerging alternative to traditional antibiotics given their broad-spectrum activity and low susceptibility to the development of resistance. To date, however, much remains unclear regarding their mechanism of action. Using functional assays (ATP leakage, cell viability, DNA binding) and super-high resolution structured illumination microscopy (OMX-SR) of fluorescently tagged polymers, we present evidence for a complex mechanism, involving membrane permeation as well as cellular uptake, interaction with intracellular targets and possible complexation with bacterial DNA. STATEMENT OF SIGNIFICANCE: This manuscript details the first study to systematically and directly investigate the mechanism of action of antimicrobial polymers, using super-resolution fluorescence imaging as well as functional assays. While many in the field cite membrane permeation as the sole mechanism underlying the activity of such polymers, we present evidence for multimodal actions including high cellular uptake and interaction with intracellular targets. It is also the first report to show competitive binding of antimicrobial polymers with bacterial DNA in a dose-dependent manner.

A microreactor with thousands of subcompartments: enzyme-loaded liposomes within polymer capsules.

Fully loaded: Noncovalent anchoring of liposomes into polymer multilayered films with cholesterol-modified polymers allows the preparation of capsosomes-liposome-compartmentalized polymer capsules (see picture). A quantitative enzymatic reaction confirmed the presence of active cargo within the capsosomes and was used to determine the number of subcompartments within this novel biomedical carrier system.

Surface modification of electrospun fibres for biomedical applications: A focus on radical polymerization methods.

The development of electrospun ultrafine fibres from biodegradable and biocompatible polymers has created exciting opportunities for biomedical applications. Fibre meshes with high surface area, suitable porosity and stiffness have been produced. Despite desirable structural and topographical properties, for most synthetic and some naturally occurring materials, the nature of the fibre surface chemistry has inhibited development. Hydrophobicity, undesirable non-specific protein adsorption and bacterial attachment and growth, coupled with a lack of surface functionality in many cases and an incomplete understanding of the myriad of interactions between cells and extracellular matrix (ECM) proteins have impeded the application of these systems. Chemical and physical treatments have been applied in order to modify or control the surface properties of electrospun fibres, with some success. Chemical modification using controlled radical polymerization, referred to here as reversible-deactivation radical polymerization (RDRP), has successfully introduced advanced surface functionality in some fibre systems. Atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT) are the most widely investigated techniques. This review analyses the practical applications of electrospinning for the fabrication of high quality ultrafine fibres and evaluates the techniques available for the surface modification of electrospun ultrafine fibres and includes a detailed focus on RDRP approaches.

Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.

Polymers fight HIV: potent (pro)drugs identified through parallel automated synthesis.

Macromolecular (pro)drugs interfere with the proliferation of HIV through both inhibition of viral cell entry and via intracellular delivery of antiviral drugs. Lead polymer conjugates exhibit longevity of action exceeding that of parent nucleoside analogue drug and are active in primary T cell over at least 72 h.

Liposomal drug deposits in poly(dopamine) coatings: effect of their composition, cell type, uptake pathway considerations, and shear stress.

Implantable devices equipped with coatings which have the ability to carry and deliver active compounds are of great interest. We report the assembly of liposome-containing poly(dopamine) films, and their interaction with adhering cells. The liposome composition is varied by adding lipophilic dopamine-conjugates and charged lipids. The cell mean fluorescence (CMF) of adhering cells due to the internalization of fluorescent cargo is found to be similar for coatings with the lipophilic-dopamine conjugates, while the charge affects the amount and location of the internalized cargo. The uptake mechanism for cargo by myoblasts using chemical inhibitors is found to be dependent on the used type of liposome. The CMF is significantly reduced for endothelial cells adhering to coatings with applied shear stress.

Highly-branched poly(N-isopropylacrylamide) as a component in poly(dopamine) films.

Mixed one-step poly(dopamine) (PDA)/highly branched poly(N-isopropylacrylamide) (pNiPAAm) coatings have been assembled and characterized by X-ray photoelectron spectroscopy (XPS), UV-vis spectroscopy, atomic force microscopy, and quartz crystal microbalance with dissipation monitoring (QCM-D) depending on the deposition temperature below and above the lower critical solution temperature (LCST) of the pNiPAAm. Mixed films were confirmed. The protein adsorption at 24 °C was found to be reduced with increasing amount of pNiPAAm in the mixed coatings, while there was no difference observed for proteins deposition at 39 °C. Further, the ability of these mixed coatings in comparison to the pure PDA and pNiPAAm films to serve as capping layer for surface-immobilized zwitterionic or positively charged liposomes has been assessed by QCM-D. The adhesion of hepatocytes, macrophages, and myoblast to these liposomes-containing hybrid coatings and the uptake of fluorescent lipids from the surface by the adhering cells depending on the capping layers were compared. The latter aspect was found to be dependent on the used capping layer and the type of liposome as carrier for the fluorescent lipid, with the highest uptake found for positive liposomes and pure pNiPAAm as capping layer. Taken together, the assembled hybrid coatings have the potential to be used as functional coatings toward surface-mediated drug delivery.

Macromolecule functionalization of disulfide-bonded polymer hydrogel capsules and cancer cell targeting.

We present a generic and versatile method for functionalization of disulfide-stabilized PMA hydrogel capsules (HCs) with macromolecules, including a number of specific antibodies to cancer cells. Functionalization was achieved by reversible addition-fragmentation chain transfer (RAFT) polymerization of poly(N-vinyl pyrrolidone) (PVPON), which introduced biorelevant heterotelechelic end groups (thiol and amine) to the polymer chain. The PVPON with heterotelechelic end groups was conjugated to the outermost layer of PMA HCs through the thiol groups and reacted with biotin via the amine groups to generate PMA/PVPON(biotin) HCs. On the basis of the high specific interaction and high affinity between biotin and avidin, and its derivates, such as NeutrAvidin (NAv), we functionalized the PMA HCs with biotinylated antibodies. We demonstrate significantly enhanced cellular binding and internalization of the antibody (Ab)-functionalized capsules compared with control human immunoglobulin (IgG)-functionalized capsules, suggesting these capsules can specifically interact with cells through antibody/antigen recognition. We anticipate that the versatility of the functionalization approach reported in this study will assist in targeted therapeutic delivery applications.

Myoblast cell interaction with polydopamine coated liposomes.

Liposomes are widely used, from biosensing to drug delivery. Their coating with polymers for stability and functionalization purposes further broadens their set of relevant properties. Poly(dopamine) (PDA), a eumelanin-like material deposited via the "self"-oxidative polymerization of dopamine at mildly basic pH, has attracted considerable interest in the past few years due to its simplicity, flexibility yet fascinating properties. Herein, we characterize the coating of different types of liposomes with PDA depending on the presence of oleoyldopamine in the lipid bilayer and the dopamine hydrochloride concentration. Further, the interaction of these coated liposomes in comparison to their uncoated counterparts with myoblast cells is assessed. Their uptake/association efficiency with these cells is determined. Further, their dose-dependent cytotoxicity with and without entrapped hydrophobic cargo (thiocoraline) is characterized. Taken together, the reported results demonstrate the potential of PDA coated liposomes as a tool in biomedical applications.