Functionalized graphene oxide mediated nucleic acid delivery

We report a simple preparation of linear polyethylenimine-grafted graphene oxide (LP-GO) conjugates and their efficacy to transfer nucleic acids into the mammalian cells. Graphene oxide (GO), with epoxy functions on its surface, was reacted with different amounts of linear polyethylenimine (lPEI), a non-toxic polymer, to obtain three different positively charged LP-GO conjugates (LP-GO-1 to LP-GO-3), capable of interacting with negatively charged nucleic acids (gel retardation assay) and transporting them efficiently into the cells. The results show that these conjugates not only exhibited considerably higher transfection efficiency but also possessed even better cell viability than lPEI. LP-GO-2, the best system in terms of transfection efficiency, showed improved buffering capacity compared to lPEI and provided sufficient stability to bound DNA against DNase I. Further, LP-GO-2 was used for the sequential delivery of GFP specific siRNA, which resulted in ∼70% suppression of the target gene expression. Intracellular trafficking using fluorescence microscopy revealed that LP-GO-2 conjugate delivered pDNA in the nucleus within 1 h of exposure. The results indicate the prospect of using these conjugates as efficient carriers of nucleic acids for future gene therapy applications.     

Functional Polyglycidol-Based Block Copolymers for DNA Complexation

Gene therapy is an attractive therapeutic method for the treatment of genetic disorders for which the efficient delivery of nucleic acids into a target cell is critical. The present study is aimed at evaluating the potential of copolymers based on linear polyglycidol to act as carriers of nucleic acids. Functional copolymers with linear polyglycidol as a non-ionic hydrophilic block and a second block bearing amine hydrochloride pendant groups were prepared using previously synthesized poly(allyl glycidyl ether)-b-polyglycidol block copolymers as precursors. The amine functionalities were introduced via highly efficient radical addition of 2-aminoethanethiol hydrochloride to the alkene side groups. The modified copolymers formed loose aggregates with strongly positive surface charge in aqueous media, stabilized by the presence of dodecyl residues at the end of the copolymer structures and the hydrogen-bonding interactions in polyglycidol segments. The copolymer aggregates were able to condense DNA into stable and compact nanosized polyplex particles through electrostatic interactions. The copolymers and the corresponding polyplexes showed low to moderate cytotoxicity on a panel of human cancer cell lines. The cell internalization evaluation demonstrated the capability of the polyplexes to successfully deliver DNA into the cancer cells.     

Application of living free radical polymerization for nucleic acid delivery

Therapeutic gene delivery can alter protein function either through the replacement of nonfunctional genes to restore cellular health or through RNA interference (RNAi) to mask mutated and harmful genes. Researchers have investigated a range of nucleic acid-based therapeutics as potential treatments for hereditary, acquired, and infectious diseases. Candidate drugs include plasmids that induce gene expression and small, interfering RNAs (siRNAs) that silence target genes. Because of their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, cationic polymers have been extensively studied for nucleic acid delivery applications, but toxicity and particle stability have limited the clinical applications of these systems. The advent of living free radical polymerization has improved the quality, control, and reproducibility of these synthesized materials. This process yields well-defined, narrowly disperse materials with designed architectures and molecular weights. As a result, researchers can study the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity, which will improve the design of next-generation vectors. In this Account, we review findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. Researchers have used robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) to engineer materials that enhance extracellular stability and cellular specificity and decrease toxicity. In addition, we discuss polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release. Finally, we describe promising materials with a range of in vivo applications from pulmonary gene delivery to DNA vaccines.     

不同浓度共聚物在保护超声介导细胞损伤和基因转染中的作用

选用三种不同的细胞系C2C12、3T3-MDEI和CHO-E为研究对象,考察了三种不同水溶性的共聚物 F127、L61和P85在超声介导细胞损伤和基因转染中的作用.结果显示,在较低浓度时,共聚物提高了超声介导的细胞损伤(P＜0.05)和基因转染(P＜0.01);在较高浓度时,F127和 P85可以保护超声介导的细胞损伤作用(P＜0.01),而L61有较高的毒性.     

Synthesis and characterization of temperature and pH-responsive pentablock copolymers

A family of amphiphilic ABCBA pentablock copolymers based on commercially available Pluronic^® F127 block copolymers and various amine containing methacrylate monomers was synthesized via Cu(I) mediated controlled radical polymerization. The block architecture and chemical composition of the pentablock copolymers were engineered to exhibit both temperature and pH responsive self-assembly by exploiting the lower critical solution temperature of the poly(ethylene oxide)/poly(propylene oxide) blocks and the polycationic property of the poly(amine methacrylate) blocks, respectively. In aqueous solutions, the pentablock copolymers formed temperature and pH-responsive micelles. Concentrated aqueous solutions of the copolymer formed a pH-responsive, thermoreversible gel phase. The controlled radical synthesis route yielded well-defined copolymers with narrow molecular weight distributions with the benefit of mild reaction conditions. Small angle X-ray scattering, laser light scattering, cryogenic transmission electron microscopy and dynamic mechanical analysis have been used to characterize the self-assembled structures of the micellar solution and gel phases of the aqueous copolymer system. These copolymers have potential applications in controlled drug delivery and non-viral gene therapy due to their tunable phase behavior and biocompatibility.     

Group transfer polymerization and its use in water based pigment dispersants and emulsion stabilizers

Group transfer polymerization (GTP) can be used to make AB diblock acrylic polymers. It provides excellent control of the structure of these polymers. With a hydrophilic B block, these polymers have been used to prepare water based emulsions, pigment dispersions, and slurries. These systems have property advantages over emulsions and dispersions made with conventional stabilizers. These advantages include increased stability, smaller particle sizes, lower viscosities, and less moisture sensitivity. The structure of the AB diblock polymer affects the properties of both the pigment dispersion and the emulsion particle. The composition, size and ratio of each block affect the overall quality. For aqueous systems, a balance of hydrophobic ‘A’ blocks and very hydrophilic ‘B’ blocks is needed for optimum properties. The hydrophobic ‘A’ blocks, which are homo or copolymers of methacrylate monomers (such as butyl or ethylhexyl methacrylate), are surface active and can associate with either pigment or emulsion polymer surfaces. The hydrophilic ‘B’ blocks, which are neutralized acid or amine containing copolymers, provide both ionic as well as steric stabilization in water-borne systems.     

Characterization and water vapor sorption property of ABA‐type triblock copolymers derived from polyimide and poly(methyl methacrylate)

The characterization of ABA‐type triblock copolymer films derived from polyimide (PI) macroinitiator and poly(methyl methacrylate) (PMMA) synthesized by atom transfer radical polymerization was investigated by focusing on different block lengths of PMMA. The hydrophobic property tends to increase with increasing PMMA content in the triblock copolymers, while the PMMA blocks enhance the charge transfer interaction between the PI segments. The water vapor sorption measurement of triblock copolymers was determined at 35 °C. The water vapor solubility of triblock copolymers tends to decrease with increasing PMMA content. In addition, linear correlations were observed between the solubility and polymer‐free volume and polymer molecular polarity in triblock copolymers as well as in other conventional polymer families. According to Zimm?Lundberg analysis, the PMMA block segments in the triblock copolymers accelerate water vapor clustering due to the high mobility of PMMA. The mobility of PMMA block segments strongly affected not only physical properties but also the water vapor solubility of the triblock copolymers. The ABA triblock copolymerization composed of PI and PMMA is one of the effective ways to improve the hydrophobic property. © 2013 Society of Chemical Industry     

Invading target cells: multifunctional polymer conjugates as therapeutic nucleic acid carriers

Polymer-based conjugates are an interesting option and challenge for the design of nano-sized drug-delivery systems, as they require advanced conjugation chemistry and precise engineering. In the case of nucleic acid therapy, non-viral carriers face several biological barriers during the delivery process, namely 1) protection of the cargo from extracellular degradation, 2) avoidance of non-specific interactions with non-targeted tissues, 3) efficient entry into the target cells, 4) intracellular trafficking to the site of action and 5) cargo release. To take on these obstacles, multifunctional conjugates can act as “smart polymers” with microenvironment-sensing dynamics to facilitate the separate delivery steps. Synthesis of defined polymer architectures with precise functionalization enables structure-activity relationships to be investigated and the integration of key functions for efficient delivery. Thus bioresponsive polymer conjugates, which are equipped with molecular devices responding to the certain microenvironments within the delivery pathway (e.g. pH, redox potential, enzymes) can be assembled. This review focuses on the modular engineering and conjugation of multifunctional polymeric structures for the utilization as “tailor-made” nucleic acid carriers.     

Novel non-ionic block copolymers tailored for interactions with phospholipids

The bulk of literature on phospholipid membrane interactions with non-ionic amphiphilic block copolymers deals with ABA triblock copolymers of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide). This is partially the result of their commercial availability. In recent years novel block copolymers have been synthesized and their interactions with phospholipids structured as Langmuir monolayers, liposomes, bilayer lipid membranes, tethered bilayers, and living cells have been studied. This review describes some new block copolymers with potential to interact with phospholipids. There is a tremendous progress in synthesis of amphiphilic block copolymers triggered by new controlled polymerization techniques as atom transfer radical polymerization or nitroxide mediated polymerization and by the possibility to ‘click’ preformed blocks together using quantitative reactions of functional endgroups. A special focus is given to novel water soluble amphiphilic triblock copolymers of poly(glycerol monomethacrylate)-b-poly(propylene oxide)-b-poly(glycerol monomethacrylate) and their interactions with phosphatidylcholine lipids. Also block copolymers containing hydrophobic blocks with perfluoroalkyl groups are discussed since they are special in a sense that their fluorophilic blocks are neither hydrophilic nor oleophilic as this is the case for conventional amphiphilic block copolymers. Experimental methods to study block copolymer–phospholipid interactions are summarized and selected results based on special experimental techniques such as isothermal titration calorimetry, infrared reflection absorption spectroscopy and ion conductance are presented. This work is intended to convey a better quantitative understanding of amphiphilic block copolymers used for in vitro and in vivo experiments in medicine and pharmacy.     

Pluronic嵌段共聚物胶束作为靶向药物载体

聚氧乙烯 聚氧丙烯 聚氧乙烯 (PEO PPO PEO)三嵌段共聚物 (商品名为Pluronics)在水溶液中能自发生成多分子聚集的胶束 ,这些胶束主要以疏水的PPO嵌段为内核 ,PEO嵌段环绕在外构成外壳 ,这种胶束可以有效地增溶油溶性药物。Pluronic嵌段共聚物无毒、无刺激、无免疫原性 ,胶束外壳的PEO嵌段能阻止血小板的聚集。胶束尺寸和病毒相仿 ,其大小适合在体内传输。初步尝试表明 ,胶束表面嵌上合适的抗体可以将增溶了模型药物的Pluronic胶束定向输送到动物脑部 ,从而提高了药效 ,降低了副作用。实验表明 ,Pluronic嵌段共聚物胶束可能成为将多种药物导向特定部位的有效载体。     

Hydrophobic and hydrophilic modifications of polyethylenimine towards gene delivery applications

Non-viral gene delivery has emerged as a promising approach for therapy in cancer treatment. Polyethylenimine (PEI) is a prominent transfecting agent, but due to toxicity and poor hemocompatibility, its usage for in vivo applications is limited. However, modification of PEI with different chemical groups can lead to conjugates with good transfection efficiency and better cytocompatibility. In this study, the efficiency of PEI derivatives, namely, PEI succinate (PEIS), PEI lauryl succinate (PEILS), PEI laurate (PEIL) was analyzed for gene delivery applications. Apart from biophysical characterization such as size, zeta potential, nanoplex stability and buffering capacity, cellular internalization, polymer trafficking and p53 transgene expression in C6 cell lines were also investigated. The results indicate that PEI conjugated with lauryl succinate act as better transfecting agent with high efficiency compared to other derivatives, and such balanced hydrophilic-hydrophobic modification of PEI can render it to be a cytocompatible and effective nucleic acid delivery system.     

DNA block copolymers: functional materials for nanoscience and biomedicine

We live in a world full of synthetic materials, and the development of new technologies builds on the design and synthesis of new chemical structures, such as polymers. Synthetic macromolecules have changed the world and currently play a major role in all aspects of daily life. Due to their tailorable properties, these materials have fueled the invention of new techniques and goods, from the yogurt cup to the car seat belts. To fulfill the requirements of modern life, polymers and their composites have become increasingly complex. One strategy for altering polymer properties is to combine different polymer segments within one polymer, known as block copolymers. The microphase separation of the individual polymer components and the resulting formation of well defined nanosized domains provide a broad range of new materials with various properties. Block copolymers facilitated the development of innovative concepts in the fields of drug delivery, nanomedicine, organic electronics, and nanoscience. Block copolymers consist exclusively of organic polymers, but researchers are increasingly interested in materials that combine synthetic materials and biomacromolecules. Although many researchers have explored the combination of proteins with organic polymers, far fewer investigations have explored nucleic acid/polymer hybrids, known as DNA block copolymers (DBCs). DNA as a polymer block provides several advantages over other biopolymers. The availability of automated synthesis offers DNA segments with nucleotide precision, which facilitates the fabrication of hybrid materials with monodisperse biopolymer blocks. The directed functionalization of modified single-stranded DNA by Watson-Crick base-pairing is another key feature of DNA block copolymers. Furthermore, the appropriate selection of DNA sequence and organic polymer gives control over the material properties and their self-assembly into supramolecular structures. The introduction of a hydrophobic polymer into DBCs in aqueous solution leads to amphiphilic micellar structures with a hydrophobic polymer core and a DNA corona. In this Account, we discuss selected examples of recent developments in the synthesis, structure manipulation and applications of DBCs. We present achievements in synthesis of DBCs and their amplification based on molecular biology techniques. We also focus on concepts involving supramolecular assemblies and the change of morphological properties by mild stimuli. Finally, we discuss future applications of DBCs. DBC micelles have served as drug-delivery vehicles, as scaffolds for chemical reactions, and as templates for the self-assembly of virus capsids. In nanoelectronics, DNA polymer hybrids can facilitate size selection and directed deposition of single-walled carbon nanotubes in field effect transistor (FET) devices.     

Nonaqueous emulsion copolymerization of ethyl methacrylate/lauryl methacrylate in propylene glycol. I. Evaluation of stabilizing efficiency for PEO‐PS‐PEO triblock copolymers

Sixteen poly(ethylene oxide)–polystyrene–poly(ethylene oxide) (PEO‐PS‐PEO) triblock copolymers were synthesized by anionic polymerization. They were characterized by gel permeation chromatography and proton NMR. The molecular weight of these 16 PEO‐PS‐PEO triblock copolymers ranged from 5100 to 13,300. The polystyrene (PS) block length was between 13 and 41. The PEO block length was between 41 and 106. The polydispersity index for these PEO‐PS‐PEO triblock copolymers were 1.05 ± 0.02. When using these stabilizers in the emulsion copolymerization of ethyl methacrylate and lauryl methacylate in propylene glycol, only a narrow window of stability was observed. Stable latexes were formed only when the molecular weights of the PEO blocks were within the range of 5300–7700 and the molecular weights of the PS blocks were 2000–4000. The stabilizer ability for these triblock copolymers was correlated with their molecular weight and conformation in propylene glycol. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 1951–1962, 2001     

ROS-sensitive selenium-containing cationic brush polymer with potent gene transfection efficiency and biocompatibility

Smart gene delivery vectors are gaining increasing attention in gene therapy, owing to their low cytotoxicity and intrinsic responsiveness. Our previously fabricated novel cationic brush polymer, comprising C Se bonds and tertiary amine EGIn-g-PDMAEMA, shows potential for gene transfection. In this study, its high efficiency for siRNA/pDNA transfection and low cytotoxicity in reactive oxygen species (ROS)-rich microenvironments is substantiated in vitro. Its superior binding capacity with siRNA/pDNA is confirmed by agarose gel electrophoresis assay. The threshold weight ratios for siRNA/pDNA migration delay are 15 and 3 (polymer-to-nucleic acid, w/w), respectively. Fluorescence microscopy and ribonucleotide reductase regulatory subunit M2 gene silencing essay verify the biodegradability and responsive control release of nucleic acids under hydrogen peroxide stimulation in Huh-7 cells. Compared with the gold standard, polyethylenimine 25 kDa, the target polymer displays superior transfection efficiency in ROS-rich tumor cells under serum-free conditions. Furthermore, the vector–nucleic acid complexes exhibit over 90% cell viability at a high concentration of 12 μg mL⁻¹ and good colloidal stability in phosphate-buffered saline (PBS) and 10% fetal bovine serum-PBS for 24 h. The efficient control release and expression of nucleic acids in ROS environments and reduced cytotoxicity highlight the superiority of EGIn-g-PDMAEMA as a gene delivery platform for tumor gene therapy.     

Nonfouling biomaterials based on polyethylene oxide‐containing amphiphilic triblock copolymers as surface modifying additives: Synthesis and characterization of copolymers and surface properties of copolymer–polyurethane blends

The objective of this work is to develop nonfouling biomaterials by blending polyethylene oxide (PEO)‐containing block copolymers with a polyurethane (PU) matrix; it is expected that the PEO component will migrate to the tissue‐material interface. Three amphiphilic triblock copolymers, PEO‐PU‐PEO, in which the PEO MW was 550 (copolymer 1), 2000 (copolymer 2), and 5000 (copolymer 3), respectively, were synthesized. XPS data showed that the polymer/vacuum interfaces of copolymers 2 and 3 were enriched in the PU block, whereas that of copolymer 1 was enriched in the PEO block. In contact with water, the PEO blocks for all three copolymers migrated to the surface as indicated by water contact angles. Blends of the copolymers with a segmented polyurethane were investigated. Surface enrichment of the copolymers occurred and increased over time up to a limit; the degree of enrichment was dependent on PEO block size and copolymer content. At copolymer content <10%, enrichment decreased with increasing PEO block size. For the copolymer 2 and copolymer 3 blends, enrichment increased with increasing copolymer content; at 20% copolymer the surfaces consisted essentially of pure copolymer. For the copolymer 1 blends, the surface was completely covered by copolymer at content ≥ 1%. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Synthesis and Characterization of Poly (phenylene oxide)-Based Block Copolymers via Cobalt Mediated Radical Polymerization (CMRP)

Novel well-defined poly(phenylene oxide) (PPO) based block copolymers were polymerized from 4-bromo-2,6-dimethylphenol (BDMP) in the presence of a cobalt acetylacetonate (Co(acac)₂) catalyst with dimethyl formamide (DMF) as a ligand and initiated with benzoyl peroxide (BPO) at 60 °C. The monomer copolymerized with vinyl acetate (VAc), polymethyl(meth)acrylate-b-poly(dimethyl siloxane)-b-polymethyl(meth)acrylate (PMMA-b-PDMS-b-PMMA) and polystyrene-b-poly(dimethyl siloxane)-b-polystyrene (PSt-b-PDMS-b-PSt) triblock copolymers in good yield. Characterization indicated a very narrow molecular weight distribution. The solid polymer obtained is a stable polymer with high thermal stability at 220 °C. The block copolymer indicated a new microstructure of phenylene oxide which was analyzed by proton-nuclear magnetic resonance (¹H-NMR), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetriy (DSC) and gel permeation chromatography (GPC). Meanwhile, the number average molecular weights calculated from the ¹H-NMR spectra were in very good agreement with the theoretically calculated values. The DSC results also indicated the successful formation of these new block copolymers.     

In vitro degradation behavior of biodegradable 4-star micelles

Drug delivery vectors for sustained release include a variety of polymeric constituents, both natural and synthetic. Among synthetic polymers several linear block copolymer systems have been explored for use as drug delivery vectors. Release of the pharmaceutical agent is affected by the degradation characteristics and/or by the swelling of the polymer. The goal of this study is to evaluate the degradation behavior of branched polyethylene oxide polylactide polyether ester as a drug delivery vector. Three samples of a star polyethylene oxide/polylactide copolymer with differing polylactide chain lengths were evaluated by characterizing the thermal properties of the neat polymer and in vitro degradation behavior.The thermal and morphological properties were examined by DSC, TGA and XRD. The in vitro polymeric micelle samples were observed over time by UV-vis, TEM and fluorescence. The four star PEO-PLA polymers have exceptional amphiphilic characteristics, which enable their use for a variety of applications. The polymers are thermally stable at biological conditions. In addition, the star polymers have shorter degradation times as compared to previously reported linear PLA and PEG-PLA copolymers, suggesting use as a short-term drug release agent. The four star PEO/PLA copolymer may be an excellent candidate for drug delivery applications.     

Synthesis and properties of copolypyromellitimides

Copolyamic acids with different proportions of diamine component were prepared by polymerizing different molar ratios of diamines—benzidine (B)/4,4′-diaminodiphenyl ether (E) and p-phenylene diamine (P)/4,4′-diaminodiphenyl methane (M)—with pyromellitic dianhydride (PMDA) in dimethylacetamide (DMAc) at room temperature. Diamine component can be arranged in regular sequence through various reaction processes, such as alternating, block, and partial block copolymers. In addition, it can also be arranged in random sequence to obtain random copolymers. Thermal cyclodehydration of polyamic acids results in the corresponding polyimides. Polymers are characterized by viscosity, thermal stability, crystallinity, and mechanical strength. It was found that an increase in the proportion of more flexible diamine component (such as E and M) incorporated in polymer chain results in copolyimides with better mechanical strength and causes a fall in viscosity of copolyamic acids and a decrease in thermal stability and crystallinity of copolyimides. Within the copolymers of the same composition, the thermal stability, crystallinity, and mechanical strength of ordered polymers are superior to those of random polymers. The results of viscosity measurements imply that the anhydride-terminated prepolymer is easily destroyed by water in the solution, so that the ultimate viscosities of alternating and block copolyamic acids are inferior to those of random ones, but this phenomenon can be improved through the preparation of the partial block copolymers.     

Poly(hydroxyether of bisphenol A) ‐alt‐polydimethylsiloxane: a novel thermally crosslinkable alternating block copolymer

BACKGROUND: An important strategy for making polymer materials with combined properties is to prepare block copolymers consisting of well‐defined blocks via facile approaches. RESULTS: Poly(hydroxyether of bisphenol A)‐block‐polydimethylsiloxane alternating block copolymers (PH‐alt‐PDMS) were synthesized via Mannich polycondensation involving phenolic hydroxyl‐terminated poly(hydroxyether of bisphenol A), diaminopropyl‐terminated polydimethylsiloxane and formaldehyde. The polymerization was carried out via the formation of benzoxazine ring linkages between poly(hydroxyether of bisphenol A) and polydimethylsiloxane blocks. Differential scanning calorimetry and small‐angle X‐ray scattering show that the alternating block copolymers are microphase‐separated. Compared to poly(hydroxyether of bisphenol A), the copolymers displayed enhanced surface hydrophobicity (dewettability). In addition, subsequent crosslinking can occur upon heating the copolymers to elevated temperatures owing to the existence of benzoxazine linkages in the microdomains of hard segments. CONCLUSION: PH‐alt‐PDMS alternating block copolymers were successfully obtained. The subsequent self‐crosslinking of the PH‐alt‐PDMS alternating block copolymers could lead to these polymer materials having potential applications. Copyright © 2008 Society of Chemical Industry     

Synthesis and study of block copolymers based on caprolactam and polyethylene glycol

High-molecular-weight block copolymers of polycaproamide and polyethylene glycol were synthesized and are distinguished by the different size of the polyamide block and number of polyamide and polyethylene oxide blocks in the macromolecule. In the conditions of synthesis of the copolymers used. their molecular characteristics are in good agreement with the calculated characteristics, and the melting point is a linear function of the reciprocal of the average degree of polymerization of the polyamide block in the copolymer. An opinion was advanced concerning the expedient regions of practical use of block copolymers differing in the ratio of stiff (polyamide) and flexible (polyethylene oxide) blocks.Ivanovo State Academy of chemical Engineering. Translated from Khimicheskie Volokna, No. 4, pp. 55–57, July–August, 1996.