Copper Nanodrugs by Atom Transfer Radical Polymerization.

In this study, some copper catalysts used for atom transfer radical polymerization (ATRP) were explored as efficient anti-tumor agents. The aqueous solution of copper-containing nanoparticles with uniform spheric morphology was in situ prepared through a copper-catalyzed activator generated by electron transfer (AGET) ATRP in water. Nanoparticles were then directly injected into tumor-bearing mice for antitumor chemotherapy. The copper nanodrugs had prolonged blood circulation time and enhanced accumulation at tumor sites, thus showing potent antitumor activity. This work provides a novel strategy for precise and large-scale preparation of copper nanodrugs with high antitumor activity.

Rational Control of Protein-Protein Interactions with Protein-ATRP-Generated Protease-Sensitive Polymer Cages.

Protease-protease interactions lie at the heart of the biological cascades that provide rapid molecular responses to living systems. Blood clotting cascades, apoptosis signaling networks, bacterial infection, and virus trafficking have all evolved to be activated and sustained by protease-protease interactions. Biomimetic strategies designed to target drugs to specific locations have generated proprotein drugs that can be activated by proteolytic cleavage to release native protein. We have previously demonstrated that the modification of enzymes with a custom-designed comb-shaped polymer nanoarmor can shield the enzyme surface and eliminate almost all protein-protein interactions. We now describe the synthesis and characterization of protease-sensitive comb-shaped nanoarmor cages using poly(ethylene glycol) methacrylate macromonomers where the PEG tines of the comb are connected to the backbone of the growing polymer chain by peptide linkers. Protease-induced cleavage of the tines of the comb releases a polymer-modified protein that can once again participate in protein-protein interactions. Atom transfer radical polymerization (ATRP) was used to copolymerize the macromonomer and carboxybetaine methacrylate from initiator-labeled chymotrypsin and trypsin enzymes, yielding proprotease conjugates that retained activity toward small peptide substrates but prevented activity against proteins. Native proteases triggered the release of the PEG side chains from the polymer backbone within 20 min, thereby increasing the activity of the conjugate toward larger protein substrates by 100%. Biomimetic cascade initiation of nanoarmored protease-sensitive protein-polymer conjugates may open the door to a new class of responsive targeted therapies.

Injectable bottlebrush hydrogels with tissue-mimetic mechanical properties.

Injectable hydrogels are desired in many biomedical applications due to their minimally invasive deployment to the body and their ability to introduce drugs. However, current injectables suffer from mechanical mismatch with tissue, fragility, water expulsion, and high viscosity. To address these issues, we design brush-like macromolecules that concurrently provide softness, firmness, strength, fluidity, and swellability. The synthesized linear-bottlebrush-linear (LBL) copolymers facilitate improved injectability as the compact conformation of bottlebrush blocks results in low solution viscosity, while the thermoresponsive linear blocks permit prompt gelation at 37°C. The resulting hydrogels mimic the deformation response of supersoft tissues such as adipose and brain while withstanding deformations of 700% and precluding water expulsion upon gelation. Given their low cytotoxicity and mild inflammation in vivo, the developed materials will have vital implications for reconstructive surgery, tissue engineering, and drug delivery applications.

Grafting Polymer Brushes by ATRP from Functionalized Poly(ether ether ketone) Microparticles.

Poly(ether ether ketone) (PEEK) is a semi-crystalline thermoplastic with excellent mechanical and chemical properties. PEEK exhibits a high degree of resistance to thermal, chemical, and bio-degradation. PEEK is used as biomaterial in the field of orthopaedic and dental implants; however, due to its intrinsic hydrophobicity and inert surface, PEEK does not effectively support bone growth. Therefore, new methods to modify PEEK's surface to improve osseointegration are key to next generation polymer implant materials. Unfortunately, PEEK is a challenging material to both modify and subsequently characterize thus stymieing efforts to improve PEEK osseointegration. In this manuscript, we demonstrate how surface-initiated atom transfer radical polymerization (SI-ATRP) can be used to modify novel PEEK microparticles (PMP). The hard core-soft shell microparticles were synthesized and characterized by DLS, ATR-IR, XPS and TEM, indicating the grafted materials increased solubility and stability in a range of solvents. The discovered surface grafted PMP can be used as compatibilizers for the polymer-tissue interface.

Molecular bottlebrush with pH-responsive cleavable bonds as a unimolecular vehicle for anticancer drug delivery.

Drug delivery systems with targeted and smart properties have emerged as an efficient strategy to overcome the challenges of cancer chemotherapy such as toxic side effects and the development of multidrug resistance. In this study, a biocompatible bottlebrush polymer poly((3-(2-bromo-2-methylpropionate)propyldimethylsilyloxy)ethyl methacrylate)-graft-poly(2-methacryloyloxyethyl phosphorylcholine) P(BIBS-EMA)-g-PMPC with pH-responsive silanol cleavable bond was designed and developed for delivery of doxorubicin. A549 cell line of human lung carcinoma was tested. The synthesized bottlebrush polymer was analyzed and characterized via Fourier transform infrared spectroscopy, FTIR, nuclear magnetic resonance spectroscopy, 1H NMR, gel permeation chromatography, GPC, dynamic laser light scattering, DLS, and static laser light scattering, SLS, techniques. The cleavage process was also precisely studied to confirm the pH-responsiveness of such bottlebrush polymers. In vitro loading and release studies of doxorubicin as a model drug were examined and the results showed a pH-dependent release manner with a twice higher release rate under cancerous tissue conditions compared to standard physiological conditions. MTT cytotoxicity assay was also performed to prove the biocompatibility of the designed polymeric platform on healthy human cells. Due to the presence of bio-inspired poly(2-methacryloyloxyethyl phosphorylcholine) side chains in the prepared bottlebrush polymer, the formed polymer-drug complex could also exhibit effective internalization into tumor cells. These facts further support the potential use of this carrier in drug delivery applications and for further in vivo studies.

Controlled Synthesis of Polyphosphazenes with Chain-Capping Agents.

N-alkyl phosphoranimines were synthesized via the Staudinger reaction of four different alkyl azides with tris(2,2,2-trifluoroethyl) phosphite. N-adamantyl, N-benzyl, N-t-butyl, and N-trityl phosphoranimines were thoroughly characterized and evaluated as chain-capping compounds in the anionic polymerization of P-tris(2,2,2-trifluoroethoxy)-N-trimethylsilyl phosphoranimine monomer. All four compounds reacted with the active chain ends in a bulk polymerization, and the alkyl end groups were identified by 1H-NMR spectroscopy. These compounds effectively controlled the molecular weight of the resulting polyphosphazenes. The chain transfer constants for the monomer and N-benzyl phosphoranimine were determined using Mayo equation.

Glycopolymer Brushes by Reversible Deactivation Radical Polymerization: Preparation, Applications, and Future Challenges.

The cellular surface contains specific proteins, also known as lectins, that are carbohydrates receptors involved in different biological events, such as cell-cell adhesion, cell recognition and cell differentiation. The synthesis of well-defined polymers containing carbohydrate units, known as glycopolymers, by reversible deactivation radical polymerization (RDRP) methods allows the development of tailor-made materials with high affinity for lectins because of their multivalent interaction. These polymers are promising candidates for the biomedical field, namely as novel diagnostic disease markers, biosensors, or carriers for tumor-targeted therapy. Although linear glycopolymers are extensively studied for lectin recognition, branched glycopolymeric structures, such as polymer brushes can establish stronger interactions with lectins. This specific glycopolymer topology can be synthesized in a bottlebrush form or grafted to/from surfaces by using RDRP methods, allowing a precise control over molecular weight, grafting density, and brush thickness. Here, the preparation and application of glycopolymer brushes is critically discussed and future research directions on this topic are suggested.

Iron Catalysts in Atom Transfer Radical Polymerization.

Catalysts are essential for mediating a controlled polymerization in atom transfer radical polymerization (ATRP). Copper-based catalysts are widely explored in ATRP and are highly efficient, leading to well-controlled polymerization of a variety of functional monomers. In addition to copper, iron-based complexes offer new opportunities in ATRP catalysis to develop environmentally friendly, less toxic, inexpensive, and abundant catalytic systems. Despite the high efficiency of iron catalysts in controlling polymerization of various monomers including methacrylates and styrene, ATRP of acrylate-based monomers by iron catalysts still remains a challenge. In this paper, we review the fundamentals and recent advances of iron-catalyzed ATRP focusing on development of ligands, catalyst design, and techniques used for iron catalysis in ATRP.

Oxygen Tolerant and Cytocompatible Iron(0)-Mediated ATRP Enables the Controlled Growth of Polymer Brushes from Mammalian Cell Cultures.

The use of zerovalent iron (Fe0)-coated plates, which act both as a source of catalyst and as a reducing agent during surface-initiated atom transfer radical polymerization (SI-ATRP), enables the controlled growth of a wide range of polymer brushes under ambient conditions utilizing either organic or aqueous reaction media. Thanks to its cytocompatibility, Fe0 SI-ATRP can be applied within cell cultures, providing a tool that can broadly and dynamically modify the substrate's affinity toward cells, without influencing their viability. Upon systematically assessing the application of Fe-based catalytic systems in the controlled grafting of polymers, Fe0 SI-ATRP emerges as an extremely versatile technique that could be applied to tune the physicochemical properties of a cell's microenvironments on biomaterials or within tissue engineering constructs.

Rapid On-Demand Extracellular Vesicle Augmentation with Versatile Oligonucleotide Tethers.

Exosomes show potential as ideal vehicles for drug delivery because of their natural role in transferring biological cargo between cells. However, current methods to engineer exosomes without negatively impacting their function remain challenging. Manipulating exosome-secreting cells is complex and time-consuming, while direct functionalization of exosome surface proteins suffers from low specificity and low efficiency. We demonstrate a rapid, versatile, and scalable method with oligonucleotide tethers to enable diverse surface functionalization on both human and murine exosomes. These exosome surface modifiers, which range from reactive functional groups and small molecules to aptamers and large proteins, can readily and efficiently enhance native exosome properties. We show that cellular uptake of exosomes can be specifically altered with a tethered AS1411 aptamer, and targeting specificity can be altered with a tethered protein. We functionalize exosomes with an immunomodulatory protein, FasL, and demonstrate their biological activity both in vitro and in vivo. FasL-functionalized exosomes, when bioprinted on a collagen matrix, allows spatial induction of apoptosis in tumor cells and, when injected in mice, suppresses proliferation of alloreactive T cells. This oligonucleotide tethering strategy is independent of the exosome source and further circumvents the need to genetically modify exosome-secreting cells.

Iron-Catalyzed Atom Transfer Radical Polymerization of Semifluorinated Methacrylates.

Fluorinated polymers are an important class of functional materials that exhibit unique properties such as high chemical resistance, thermal stability, and low surface energy. Atom transfer radical polymerization (ATRP) of semifluorinated monomers catalyzed by copper catalysts often requires development of special conditions to control the polymerization and prevent side reactions such as base-catalyzed transesterification between the fluoro-containing monomers and solvents. In this paper, photoinduced iron-catalyzed ATRP was applied to the polymerization of a variety of semifluorinated methacrylate monomers. Polymerizations were initiated by photochemical generation of the Fe catalyst activator under blue light irradiation, enabling temporal control over the growth of polymer chains, and were well-controlled in various solvents, including fluorinated and nonfluorinated solvents, without undergoing any side reactions. Moreover, in situ chain extension and block copolymerization experiments demonstrated the preservation of chain end functionality, enabling facile synthesis of well-controlled block copolymers.

Tailoring Site Specificity of Bioconjugation Using Step-Wise Atom-Transfer Radical Polymerization on Proteins.

Protein-polymer conjugates are powerful combinations of the biotic and abiotic worlds that impact many industries. Predicting the site and impact of polymer growth from the surface of proteins is only useful if we can use that information to choose which site to modify synthetically. We have explored the combination of a predictive algorithm with a unique stepwise atom-transfer radical polymerization (ATRP) to selectively move the predominant modification sites around a model enzyme. Lysozyme was modified with defined stoichiometric ratios of polymerization initiators and initiation inhibitors to selectively and strategically grow poly(carboxybetaine methacrylate) polymers from different protein sites. Electrospray ionization mass spectrometry was used to examine the uniformity of the lysozyme-initiator and lysozyme-inhibitor complexes prior to polymer growth. Bioactivity of the lysozyme-polymer conjugates was examined as a function of polymer location on the enzyme surface. Step-wise atom-transfer radical polymerization from proteins provides a versatile and modular approach that can be extended to the rational and selective design of other protein-polymer conjugates.

Cationic Hyperbranched Polymers with Biocompatible Shells for siRNA Delivery.

Cationic hyperbranched polymers (HBP) were prepared by self-condensing vinyl polymerization of an atom transfer radical polymerization (ATRP) inimer containing a quaternary ammonium group. Two types of biocompatible shells, poly(oligoethylene glycol) methacrylate (polyOEGMA) and poly(2-(methylsulfinyl) ethyl methacrylate) (polyDMSO), were grafted respectively from HBP core to form core-shell structures with low molecular weight dispersity and high biocompatibility, polyOEGMA-HBP and polyDMSO-HBP. Both of the structures showed low cytotoxicity and good siRNA complexing ability. The efficacy of gene silencing against Runt-related transcription factor 2 ( Runx2) expression and the long-term assessment of mineralized nodule formation in osteoblast cultures were evaluated. The biocompatible core-shell structures were crucial to minimizing undesired cytotoxicity and nonspecific gene suppression. polyDMSO-HBP showed higher efficacy of forming polyplexes than polyOEGMA-HBP due to shell with lower steric hindrance. Overall, the gene silencing efficiency of both core-shell structures was comparable to commercial agent Lipofectamine, indicating long-term potential for gene silencing to treat heterotopic ossification (HO).

Direct ATRP of Methacrylic Acid with Iron-Porphyrin Based Catalysts.

An iron porphyrin catalyst, derived from the active center of proteins such as horseradish peroxidase and hemoglobin, was successfully used for the atom transfer radical polymerizations (ATRP) of methacrylic acid. ATRP of methacrylic acid and other acidic monomers is challenging due to Cu complexation by carboxylates, protonation of the ligand, and displacement of the halogen chain end. A robust mesohemin-based catalyst provided controlled ATRP of methacrylic acid, yielding poly(methacrylic acid) with Mn ≥ 20000 and dispersity D < 1.5. Retention of halogen chain end was confirmed by successful chain extension of a poly-(methacrylic acid)-Br macroinitiator.

Iron Oxide Nanoparticles with Grafted Polymeric Analogue of Dimethyl Sulfoxide as Potential Magnetic Resonance Imaging Contrast Agents.

Novel water-dispersible hybrid iron oxide nanoparticles grafted with a polymeric analogue of dimethyl sulfoxide (DMSO) were prepared. Superparamagnetic iron oxide nanoparticles with immobilized atom-transfer radical polymerization (ATRP) initiators were prepared via an in situ method using 12-(2-bromoisobutyramido)dodecanoic acid as a surface ligand/initiator. The initiator-functionalized particles were employed in a surface-initiated initiator for continuous activator regeneration ATRP to graft poly(2-(methylsulfinyl)ethyl acrylate) (a polyacrylate analogue of DMSO) from the surface. The resulting hybrid nanoparticles showed a high magnetic relaxivity ratio ( r2/ r1) of 600 at 7 T in fetal bovine serum, and a good biocompatibility up to 1000 mg L-1.

Solid-phase synthesis of protein-polymers on reversible immobilization supports.

Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

Enhanced interfacial activity of multi-arm poly(ethylene oxide) star polymers relative to linear poly(ethylene oxide) at fluid interfaces.

Interfacial tension reduction, dynamic dilatational elasticity and extent of adsorption were investigated for linear poly(ethylene oxide) (PEO) chains of varying molecular weight and for PEO star polymers with an average of 64 arms per star at air/water, xylene/water, and cyclohexane/water interfaces. Adsorption on planar interfaces was monitored by ellipsometry, while interfacial tension and dilatational elasticity were measured separately by pendant drop tensiometry. Previously reported to be efficient emulsifiers, PEO stars are shown here to also be more effective foaming agents than linear PEO. Accordingly, PEO stars adsorb to a greater extent and produce larger interfacial tension reduction and greater dynamic dilatational moduli than linear PEO. The more extensive adsorption and greater interfacial tension reduction for PEO stars are attributed to their compactness. More mass is introduced per unit area of interface, and more interfacial penetration is achieved, upon their adsorption than for adsorption of linear polymers that adopt the conformation of loops, trains and tails. Whereas cyclohexane is a non-solvent for PEO, xylene is a good solvent. Dispersing PEO stars in the xylene phase yields greater interfacial tension reduction at the xylene/water interface than occurs when initially dispersing PEO stars in the aqueous phase. In contrast, the interfacial tension for linear PEO shows no dependence on the phase from which it adsorbs. Ellipsometry confirms the path-dependent extent of adsorption to the xylene/water interface, but also reveals additional complexity. When adsorbing from xylene, thick interfacial films result that likely contain dispersed water, as suggested by the observation of spontaneous water-in-xylene emulsification when PEO stars are initially dispersed in xylene. This is tentatively attributed to shear provided by Marangoni flow. Spontaneous emulsification occurs only when PEO stars are initially dispersed in the xylene phase. Linear PEO produces neither thick interfacial films nor spontaneous emulsification.

A brush-polymer conjugate of exendin-4 reduces blood glucose for up to five days and eliminates poly(ethylene glycol) antigenicity.

The delivery of therapeutic peptides and proteins is often challenged by a short half-life, and thus the need for frequent injections that limit efficacy, reduce patient compliance and increase treatment cost. Here, we demonstrate that a single subcutaneous injection of site-specific (C-terminal) conjugates of exendin-4 (exendin) - a therapeutic peptide that is clinically used to treat type 2 diabetes - and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) with precisely controlled molecular weights lowered blood glucose for up to 120 h in fed mice. Most notably, we show that an exendin-C-POEGMA conjugate with an average of 9 side-chain ethylene glycol (EG) repeats exhibits significantly lower reactivity towards patient-derived anti-poly(ethylene glycol) (PEG) antibodies than two FDA-approved PEGylated drugs, and that reducing the side-chain length to 3 EG repeats completely eliminates PEG antigenicity without compromising in vivo efficacy. Our findings establish the site-specific conjugation of POEGMA as a next-generation PEGylation technology for improving the pharmacological performance of traditional PEGylated drugs, whose safety and efficacy are hindered by pre-existing anti-PEG antibodies in patients.

In Vivo GFP Knockdown by Cationic Nanogel-siRNA Polyplexes.

RNA interference (RNAi) is a powerful tool to treat diseases and elucidate target gene function. Prior to clinical implementation, however, challenges including the safe, efficient and targeted delivery of siRNA must be addressed. Here, we report cationic nanogel nanostructured polymers (NSPs) prepared by atom transfer radical polymerization (ATRP) for in vitro and in vivo siRNA delivery in mammalian models. Outcomes from siRNA protection studies suggested that nanogel NSPs reduce enzymatic degradation of siRNA within polyplexes. Further, the methylation of siRNA may enhance nuclease resistance without compromising gene knockdown potency. NSP-mediated RNAi treatments against Gapdh significantly reduced GAPDH enzyme activity in mammalian cell culture models supplemented with 10% serum. Moreover, nanogel NSP-mediated siRNA delivery significantly inhibited in vivo GFP expression in a mouse model. GFP knockdown was siRNA sequence-dependent and facilitated by nanogel NSP carriers. Continued testing of NSP/siRNA compositions in disease models may produce important new therapeutic options for patient care.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells.

BACKGROUND: Heterotopic ossification (HO) may occur after musculoskeletal trauma, traumatic brain injury, and total joint arthroplasty. As such, HO is a compelling clinical concern in both military and civilian medicine. A possible etiology of HO involves dysregulated signals in the bone morphogenetic protein osteogenic cascade. Contemporary treatment options for HO (ie, nonsteroidal antiinflammatory drugs and radiation therapy) have adverse effects associated with their use and are not biologically engineered to abrogate the molecular mechanisms that govern osteogenic differentiation. QUESTIONS/PURPOSES: We hypothesized that (1) nanogel-mediated short interfering RNA (siRNA) delivery against Runt-related transcription factor 2 (Runx2) and osterix (Osx) genes will decrease messenger RNA expression; (2) inhibit activity of the osteogenic marker alkaline phosphatase (ALP); and (3) inhibit hydroxyapatite (HA) deposition in osteoblast cell cultures. METHODS: Nanogel nanostructured polymers delivered siRNA in 48-hour treatment cycles against master osteogenic regulators, Runx2 and Osx, in murine calvarial preosteoblasts (MC3T3-E1.4) stimulated for osteogenic differentiation by recombinant human bone morphogenetic protein (rhBMP-2). The efficacy of RNA interference (RNAi) therapeutics was determined by quantitation of messenger RNA knockdown (by quantitative reverse transcription-polymerase chain reaction), downstream protein knockdown (determined ALP enzymatic activity assay), and HA deposition (determined by OsteoImage™ assay). RESULTS: Gene expression assays demonstrated that nanogel-based RNAi treatments at 1:1 and 5:1 nanogel:short interfering RNA weight ratios reduced Runx2 expression by 48.59% ± 19.53% (p < 0.001) and 43.22% ± 18.01% (both p < 0.001). The same 1:1 and 5:1 treatments against both Runx2 and Osx reduced expression of Osx by 51.65% ± 10.85% and 47.65% ± 9.80% (both p < 0.001). Moreover, repeated 48-hour RNAi treatment cycles against Runx2 and Osx rhBMP-2 administration reduced ALP activity after 4 and 7 days. ALP reductions after 4 days in culture by nanogel 5:1 and 10:1 RNAi treatments were 32.4% ± 12.0% and 33.6% ± 13.8% (both p < 0.001). After 7 days in culture, nanogel 1:1 and 5:1 RNAi treatments produced 35.9% ± 14.0% and 47.7% ± 3.2% reductions in ALP activity. Osteoblast mineralization data after 21 days suggested that nanogel 1:1, 5:1, and 10:1 RNAi treatments decreased mineralization (ie, HA deposition) from cultures treated only with rhBMP-2 (p < 0.001). However, despite RNAi attack on Runx2 and Osx, HA deposition levels remained greater than non-rhBMP-2-treated cell cultures. CONCLUSIONS: Although mRNA and protein knockdown were confirmed as a result of RNAi treatments against Runx2 and Osx, complete elimination of mineralization processes was not achieved. RNAi targeting mid- and late-stage osteoblast differentiation markers such as ALP, osteocalcin, osteopontin, and bone sialoprotein) may produce the desired RNAi-nanogel nanostructured polymer HO prophylaxis. CLINICAL RELEVANCE: Successful HO prophylaxis should target and silence osteogenic markers critical for heterotopic bone formation processes. The identification of such markers, beyond RUNX2 and OSX, may enhance the effectiveness of RNAi prophylaxes for HO.