A microfluidic platform for integrated synthesis and dynamic light scattering measurement of block copolymer micelles

Microfluidic devices were developed that integrate the synthesis of well defined block copolymers and dynamic light scattering (DLS) measurement of their micelle formation. These metal devices were designed to operate in contact with organic solvents and elevated temperatures for long periods, and thus were capable of continuous in-channel atom transfer radical polymerization (ATRP) of styrene and (meth)acrylate homopolymers and block copolymers. These devices were equipped with a miniaturized fiber optic DLS probe that included several technology improvements, including a measurement volume of only 4 microlitres, simple alignment, and reduced multiple scattering. To demonstrate the integrated measurement, poly(methyl methacrylate-b-lauryl methacrylate) and poly(methyl methacrylate-b-octadecyl methacrylate) block copolymers were processed on the device with a selective solvent, dodecane, to induce micelle formation. The in situ DLS measurements yielded the size and aggregation behavior of the micelles. For example, the block copolymer solutions formed discrete micelles (D(H) approximately = 25 nm) when the corona block was sufficiently long (f(MMA) < 0.51), but the micelles aggregated when this block was short. This study demonstrates the utility of these new devices for screening the solution behavior of custom synthesized polymeric surfactants and additives.     

Synthesis of water-soluble polymeric prodrugs possessing 4-methylcatechol derivatives by mechanochemical solid-state copolymerization and nature of drug release

In this study we synthesized the water-soluble polymeric prodrugs possessing a 4-methylcatechol (4MC) derivative as a side chain by mechanochemical solid-state copolymerization. 1-benzoyl-4-methylcatechol (Bz4MC) was selected as a model compound of 4MC, and its methacryloyl derivative (1) was synthesized. 6-O-methacryloyl-D-galactose (2) was also prepared as a water-soluble monomer. The mechanochemical solid-state copolymerization of 1 and 2 was carried out to obtain the water-soluble polymeric prodrug possessing the Bz4MC as a side chain. The mechanochemical copolymerization of 1 and 2 proceeded to completion, and the polymeric prodrug produced possessed a narrow molecular weight distribution. Three kinds of polymeric prodrugs, whose compositions were different from one another, were hydrolyzed in vitro. The hydrolysis of these polymeric prodrugs proceeded to completion. The rate constants of hydrolysis decreased with increasing the mole fraction of 1 in polymeric prodrug. It was suggested that the rate constant of hydrolysis could be controlled by the composition, the mole fraction of 1 in the polymeric prodrug.     

Non-surface activity and micellization behavior of cationic amphiphilic block copolymer synthesized by reversible addition-fragmentation chain transfer process

Cationic amphiphilic diblock copolymers of poly(n-butylacrylate)-b-poly(3-(methacryloylamino)propyl)trimethylammonium chloride) (PBA-b-PMAPTAC) with various hydrophobic and hydrophilic chain lengths were synthesized by a reversible addition-fragmentation chain transfer (RAFT) process. Their molecular characteristics such as surface activity/nonactivity were investigated by surface tension measurements and foam formation observation. Their micelle formation behavior and micelle structure were investigated by fluorescence probe technique, static and dynamic light scattering (SLS and DLS), etc., as a function of hydrophilic and hydrophobic chain lengths. The block copolymers were found to be non-surface active because the surface tension of the aqueous solutions did not change with increasing polymer concentration. Critical micelle concentration (cmc) of the polymers could be determined by fluorescence and SLS measurements, which means that these polymers form micelles in bulk solution, although they were non-surface active. Above the cmc, the large blue shift of the emission maximum of N-phenyl-1-naphthylamine (NPN) probe and the low micropolarity value of the pyrene probe in polymer solution indicate the core of the micelle is nonpolar in nature. Also, the high value of the relative intensity of the NPN probe and the fluorescence anisotropy of the 1,6-diphenyl-1,3,5-hexatriene (DPH) probe indicated that the core of the micelle is highly viscous in nature. DLS was used to measure the average hydrodynamic radii and size distribution of the copolymer micelles. The copolymer with the longest PBA block had the poorest water solubility and consequently formed micelles with larger size while having a lower cmc. The "non-surface activity" was confirmed for cationic amphiphilic diblock copolymers in addition to anionic ones studied previously, indicating the universality of non-surface activity nature.     

Physicochemical characterization of degradable thermosensitive polymeric micelles

Amphiphilic AB block copolymers consisting of thermosensitive poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol), pHPMAmDL-b-PEG, were synthesized via a macroinitiator route. Dynamic light scattering measurements showed that these block copolymers form polymeric micelles in water with a size of around 50 nm by heating of an aqueous polymer solution from below to above the critical micelle temperature (cmt). The critical micelle concentration as well as the cmt decreased with increasing pHPMAmDL block lengths, which can be attributed to the greater hydrophobicity of the thermosensitive block with increasing molecular weight. Cryogenic transmission electron microscopy analysis revealed that the micelles have a spherical shape with a narrow size distribution. 1H NMR measurements in D2O showed that the intensity of the peaks of the protons from the pHPMAmDL block significantly decreased above the cmt, indicating that the thermosensitive blocks indeed form the solidlike core of the micelles. Static light scattering measurements demonstrated that pHPMAmDL-b-PEG micelles with relatively large pHPMAmDL blocks possess a highly packed core that is stabilized by a dense layer of swollen PEG chains. FT-IR analysis indicated that dehydration of amide bonds in the pHPMAmDL block occurs when the polymer dissolved in water is heated from below to above its cmt. The micelles were stable when an aqueous solution of micelles was incubated at 37 degrees C and at pH 5.0, where the hydrolysis rate of lactate side groups is minimized. On the other hand, at pH 9.0, where hydrolysis of the lactic acid side groups occurs, the micelles started to swell after 1.5 h of incubation and complete dissolution of micelles was observed after 4 h as a result of hydrophilization of the thermosensitive block. Fluorescence spectroscopy measurements with pyrene loaded in the hydrophobic core of the micelles showed that when these micelles were incubated at pH 8.6 and at 37 degrees C the microenvironment of pyrene became increasingly hydrated in time during this swelling phase. The results demonstrate the potential applicability of pHPMAmDL-b-PEG block copolymer micelles for the controlled delivery of hydrophobic drugs.     

Characterization of novel pH-sensitive polymeric micelles prepared by the self-assembly of amphiphilic block copolymer with poly-4-vinylpyridine block synthesized by mechanochemical solid-state polymerization

We fabricated novel pH-sensitive polymeric micelles consisting of amphiphilic block copolymer containing pyridyl groups as side chains in the hydrophobic block. The number average particle diameter of the polymeric micelles at pH 7 was approximately 200 nm. A decrease in pH resulted in deformation of the polymeric micelles over a very narrow pH range (between pH 5.7 and 5.6). Interestingly, micellization and demicellization occurred reversibly in this narrow pH range. Polymeric micelles incorporating 5-fluorouracil (5FU) were also prepared. Decreasing the pH of this polymeric micelle solution from 7 to 5.5 resulted in the rapid release of 5FU at pH 5.6; the drug was completely released within 30 min. These results suggest that deformation of the polymeric micelles caused the rapid release of 5FU.     

Bioinspired core‐crosslinked micelles from thymine‐functionalized amphiphilic block copolymers: Hydrogen bonding and photo‐crosslinking study

Bioinspired core‐bound polymeric micelles, based on hydrogen bonding and photo‐crosslinking, of thymine have been prepared from poly(vinylbenzylthymine)‐b‐poly(vinylbenzyltriethylammonium chloride). The amphiphilic block copolymer was synthesized by 2,2‐tetramethylpiperidin‐1‐oxyl‐mediated living radical polymerization in water/ethylene glycol solution. Micelle characterization and critical micelle concentration measurements demonstrated that the hydrogen bonding of the attached thymine units stabilizes the micelles. Further, core‐crosslinked polymeric micelles were formed by ultraviolet (UV) radiation showing that the stability of the micelle could be controlled by the UV crosslinking of the attached thymines. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Novel thermo‐responsive self‐assembly micelles from a double brush‐shaped PNIPAM‐g‐(PA‐b‐PEG‐b‐PA)‐g‐PNIPAM block copolymer with PNIPAM polymers as side chains

A novel double brush‐shaped copolymer with amphiphilic polyacrylate‐b‐poly(ethylene glycol)‐b‐poly acrylate copolymer (PA‐b‐PEG‐b‐PA) as a backbone and thermosensitive poly(N‐isopropylacrylamide) (PNIPAM) long side chains at both ends of the PEG was synthesized via an atom transfer radical polymerization (ATRP) route, and the structure was confirmed by FTIR, ¹H NMR, and SEC. The thermosensitive self‐assembly behavior was examined via UV‐vis, TEM, DLS, and surface tension measurements, etc. The self‐assembled micelles, with low critical solution temperatures (LCST) of 34–38 ^°C, form irregular fusiform and/or spherical morphologies with single, double, and petaling cores in aqueous solution at room temperature, while above the LCST the micelles took on more regular and smooth spherical shapes with diameter ranges from 45 to 100 nm. The micelle exhibits high stabilities even in simulated physiological media, with low critical micellization concentration (CMC) up to 5.50, 4.89, and 5.05 mg L^?1 in aqueous solution, pH 1.4 and 7.4 PBS solutions, respectively. The TEM and DLS determination reveled that the copolymer micelle had broad size distribution below its LCST while it produces narrow and homogeneous size above the LCST. The cytotoxicity was investigated by MTT assays to elucidate the application potential of the as‐prepared block polymer brushes as drug controlled release vehicles. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Synthesis of graft copolymer of ethyl cellulose through living polymerization and its self-assembly

Copolymers of ethyl cellulose (EC) with polystyrene (PSt) were synthesized through atom transfer radical polymerization (ATRP). The molecular weight of graft copolymers increased without any trace of the EC macro-initiator, and the polydispersity of the side chains was low. The molecular weight of the side chains increased with the monomer conversion. Kinetic study indicated that the polymerization was first order. The micelle characteristics of the graft copolymer in acetone were investigated using dynamic light scattering (DLS), atom force microscopy (AFM) and transmission electron microscopy (TEM). With increasing the concentration, micelles were gradually formed from the solution. The TEM and AFM images indicated that the micelles had spherical shape and showed core-shell structure.     

温度敏感性PNIPAM-b-PZLL-b-mPEG三嵌段共聚物的合成与自组装研究

通过大分子引发剂引发ε-苄氧羰基-L-赖氨酸-N-羧酸酐(Lys-NCA)开环聚合和大分子缩合的方法合成了聚(N-异丙基丙烯酰胺)-b-聚(ε-苄氧羰基-L-赖氨酸)-b-聚乙二醇单甲醚三嵌段共聚物(PNIPAM-b-PZLL-b-mPEG).用GPC和1H-NMR对其结构进行了表征.用芘荧光探针法证明了该三嵌段聚合物形成胶束的性质并测定了临界胶束浓度(CMC).动态光散射(DLS)研究表明,在固定PNIPAM-b-PZLL链段长度的情况下,mPEG分子量为2000时,胶束在温度高于临界溶解温度(LCST)时发生聚集,mPEG分子量为5000时,胶束在LCST以上没有发生聚集.     

Physicochemical characterization of polymeric micelles constructed from novel amphiphilic polyphosphazene with poly(N-isopropylacrylamide) and ethyl 4-aminobenzoate as side groups

An amphiphilic graft polyphosphazene (PNIPAm/EAB-PPP) composed of oligo-poly(N-isopropylacrylamide) (PNIPAm) as hydrophilic segments and ethyl 4-aminobenzoate (EAB) as hydrophobic groups was synthesized via ring-opening polymerization and subsequent substitution reaction. The molar ratio of the PNIPAm segment to EAB group was 1.85:0.15. The lower critical solution temperature (LCST) of copolymer was 32.6 degrees C as determined by turbidity method. Micellization behavior of PNIPAm/EAB-PPP in an aqueous phase was characterized by fluorescence technique, 1H NMR, dynamic light scattering (DLS) and transmission electron microscopy (TEM). The critical micelle concentration (CMC) of the graft copolymer in aqueous solution was 0.1mg/ml. The number-averaged particle size of spherical micelles was 80 nm at 25 degrees C with a narrow distribution. TEM also revealed that inter-micellar aggregation was induced in the micelle solution at temperature above LCST of graft copolymer. The thermosensitive PNIPAm/EAB-PPP micelles may be of help to regulate the loading and release of hydrophobic drugs.     

ARGET ATRP 与ROP 结合制备pH 响应两亲性聚合物分子刷及其自组装研究

结合电子转移活化剂再生-原子转移自由基聚合(ARGET ATRP)和开环聚合(ROP)法合成了一种具有无规疏水/ pH 响应结构的两亲性聚合物分子刷聚(甲基丙烯酸聚丙交酯酯-co-甲基丙烯酸)-b-聚甲基丙烯酸单甲氧基聚乙二醇酯 [P(PLAMA-co-MAA)-b-PPEGMA]. 通过核磁共振氢谱(¹H NMR)和凝胶渗透色谱(GPC)表征了聚合物的结构、分子量及分子量分布. 优化了反应条件并合成出分子量可控、分子量分布窄的聚合产物. 采用动态光散射法(DLS)、扫描电子显微镜(SEM)研究了聚合物分子刷在水溶液中自组装胶束的粒径、形貌及pH 响应行为. P(PLAMA-co-MAA)-b-PPEGMA 自组装形成粒径分布均匀的球形胶束. 且随着溶液pH 值从7 降低至3, 胶束中的PMAA 逐渐去离子化, 溶胀的胶束逐渐收缩, 粒径由200～300 nm 减小至150 nm 左右; 但当pH 值减小到2 以下, 胶束表面电荷量非常小, 胶束聚集, 使得粒径增大.     

Dynamic and static light scattering studies on self-aggregation behavior of biodegradable amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) triblock copolymers in aqueous solution

The self-aggregation behavior of two amphiphilic poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) triblock copolymer samples with nearly identical PHB block lengths but different PEO block lengths, PEO-PHB-PEO(2000-810-2000) and PEO-PHB-PEO(5000-780-5000), was studied with dynamic and static light scattering (DLS and SLS), in combination with fluorescence spectroscopy and transmission electron microscopy (TEM). The formation of polymeric micelles by the two PEO-PHB-PEO triblock copolymers was confirmed with fluorescence technique and TEM. DLS analysis showed that the hydrodynamic radius (R(h)) of the monodistributed polymeric micelles increased with an increase in PEO block length. The relative thermostability of the triblock copolymer micelles was studied by SLS and DLS at different temperatures. The aggregation number and the ratio of the radius of gyration over hydrodynamic radius were found to be independent of temperature, probably due to the strong hydrophobicity of the PHB block. The combination of DLS and SLS studies indicated that the polymeric micelles were composed of a densely packed core of hydrophobic PHB blocks and a corona shell formed by hydrophilic PEO blocks. The aggregation numbers were found to be approximately 53 for PEO-PHB-PEO(2000-810-2000) micelles and approximately 37 for PEO-PHB-PEO(5000-780-5000) micelles. The morphology of PEO-PHB-PEO spherical micelles determined by DLS and SLS measurements was further confirmed by TEM.     

嵌段共聚物聚(异丙基丙烯酰胺)-b-聚(双丙酮丙烯酰胺)制备及其对叶酸的载负和释放

以N-异丙基丙烯酰胺(NIPAm)和双丙酮丙烯酰胺(DAAM)为原料,采用可逆加成 断裂链转移(RAFT)可控聚合反应法合成了两亲性两嵌段共聚物 聚(异丙基丙烯酰胺)-b-聚(双丙酮丙烯酰胺)(PNIPAm-b-PDAAM),用红外光谱(FT-IR)、核磁共振(1H NMR)和凝胶渗透色谱(GPC)对其结构和组成进行了表征。 这种共聚物在水溶液中能够自组装成稳定的聚合物胶束,通过荧光探针测得其低临界胶束浓度(CMC)约为7.0 mg/L。 采用扫描电子显微镜(SEM)和动态激光光散射(DLS)测得,PNIPAm-b-PDAAM在水溶液中自组装成核壳结构的球形胶束,SEM测得其直径约150 nm,且分散性良好。 以其聚合物胶束为载体、叶酸(FA)为模型药物,模拟人体生理环境进行药物体外释放。 结果表明,叶酸的负载量及负载率分别为25%和74%。 在人体温度37℃、pH值分别为4.0、6.86、9.18磷酸缓冲溶液(PBS)中,FA在20 h内的释放均比25 ℃快,释放速率随pH值增加而增大,最大累积释放率分别为31%、67%和72%。     

两亲性壳聚糖衍生物的制备及其包覆量子点

以天然壳聚糖为功能性高分子的骨架,通过西弗碱还原法,以正辛醛和端醛基聚乙二醇单甲基醚(mPEG aldehyde,mPEG-ald)修饰壳聚糖,制备了具有亲疏水性质的N-辛基化-N-mPEG化壳聚糖衍生物(N-octyl-N-mPEG-chitosan,OPEGC),并且以此两亲性壳聚糖衍生物包覆量子点制备了水溶性聚合物量子点纳米粒子.用FTIR、1H-NMR对壳聚糖衍生物进行了结构表征.制备的水溶性胶束,用动态光散射(DLS)测试其流体力学直径与分布,研究了烷基链接枝比率对粒径大小的影响,研究结果表明:烷基链接枝率越高,聚合物胶束粒径越小.以芘为分子探针,通过荧光光谱法测定了壳聚糖衍生物的临界胶束浓度(CMC)为2.032×10-2mg/mL,并对水溶性聚合物量子点纳米粒子进行了紫外、荧光及形貌表征,结果表明得到了荧光发射产率高的聚合物量子点纳米粒子,且尺寸均一、水溶性好.     

Formation of reversible shell cross-linked micelles from the biodegradable amphiphilic diblock copolymer poly(L-cysteine)-block-poly(L-lactide)

A novel biodegradable diblock copolymer, poly(L-cysteine)-b-poly(L-lactide) (PLC-b-PLLA), was synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of beta-benzyloxycarbonyl-L-cysteine (ZLC-NCA) with amino-terminated poly(L-lactide) (NH 2-PLLA) as a macroinitiator in a convenient way. The diblock copolymer and its precursor were characterized by (1)H NMR, Fourier transform infrared (FT-IR), gel permeation chromatography (GPC), and X-ray photoelectron spectroscopy (XPS) measurements. The length of each block polymer could be tailored by molecular design and the ratios of feeding monomers. The cell adhesion and cell spread on the PZLC-b-PLLA and PLC-b-PLLA films were enhanced compared to those on pure PLA film. PLC-b-PLLA can self-assemble to form micelles in aqueous media. A pyrene probe is used to demonstrate the micelle formation of PLC-b-PLLA in aqueous solution. Due to the ease of disulfide exchange with thiols, the obtained micelles are reversible shell cross-linked (SCL) micelles. The morphology and size of the micelles are studied by dynamic light scattering (DLS) and environmental scanning electron microscopy (ESEM).     

Fabrication of ordered catalytically active nanoparticles derived from block copolymer micelle templates for controllable synthesis of single-walled carbon nanotubes

We report the use of the block copolymer micelle approach to produce various transition metal nanoparticles such as iron, cobalt, and nickel with precisely controlled size and spacing. These uniformly sized catalyst nanoparticles derived from the block copolymer micelle approach have enabled the synthesis of carbon nanotubes (CNTs) with narrow size distribution. Because of the excellent film forming ability of the polymeric material, metal-bearing surface micelles produced from the solution micelles can be distributed uniformly on a surface, resulting in evenly dispersed catalyst nanoparticles. As a result, high quality and uniformly distributed CNTs have been synthesized. Spatially selective growth of CNTs from a lithographically patterned metal-bearing micelle film has been achieved. The polymer template approach can potentially be extended to synthesize single-metallic and bimetallic catalytically active nanoparticles with uniform size and spacing and is fully compatible with conventional lithographic process. Additionally, catalyst nanoparticles produced from this method do not coalesce at high growth temperature. All these attributes make this approach a promising fabrication pathway for controllable synthesis of CNTs.     

pH-induced micelle formation of poly(histidine-co-phenylalanine)-block-poly(ethylene glycol) in aqueous media

Poly[(L-histidine)-co-(L-phenylalanine)]-block-poly(ethylene glycol) (HF-b-PEG) diblock copolymers were synthesized to be used for preparation of pH-sensitive polymeric micelles. First, HF block was synthesized by ring opening copolymerization of L-histidine and L-phenylalanine N-carboxyanhydride, and then the resulting copolymer was coupled with PEG. The pKa value of diblock copolymer can be controlled by adjusting the histidine/phenylalanine ratio in HF block. It is observed that the block copolymers form micelles in aqueous media and that the micelles are spherical in shape with a unimodal distribution. The micelle is formed at pH higher than pKa of block copolymer while it is not formed at lower pH. This is because the protonation of histidine residue in the HF block converts the hydrophobic core into hydrophilic one at lower pH. Acid-Base titration profile of HF41(5600)-b-PEG, HF56(5500)-b-PEG, H(5100)-b-PEG and 0.1 N NaCl.     

Surfactant‐free synthesis of amphiphilic diblock copolymer in aqueous phase by a self‐stability process

Amphiphilic polymeric particles with hydrophobic cores and hydrophilic shells were prepared via living radical emulsion polymerization of styrene using a water‐soluble poly(acrylamide)‐based macro‐RAFT agent in aqueous solution in the absence of any surfactants. Firstly, the homopolymerization of acrylamide (AM) was carried out in aqueous phase by reversible addition‐fragmentation chain transfer radical polymerization (RAFT) using a trithiocarbonate as a chain transfer agent. Then the PAM‐based macro‐RAFT agent has been used as a water‐soluble macromolecular chain transfer agent in the batch emulsion polymerization of Styrene (St) free of surfactants. The RAFT controlled growth of hydrophobic block led to the formation of well‐defined poly(acrylamide)‐copolystyrene amphiphilic copolymer, which was able to work as a polymeric stabilizer (self‐stability). Finally, very stable latex was prepared, having no visible phase separation for several months. FTIR and ¹H‐NMR measurements showed that the product was the block copolymer PAM‐co‐PS in the form of stable latex. Atomic force microscope (AFM), transmission electron microscope (TEM), and dynamic light scattering (DLS) studies indicated that the nanoparticles have a narrow particle size distribution and the average particle hydrodynamic radius was kept in the diameter of 58 nm. Core‐shell structure of the copolymer was also recorded by TEM. The mechanism of the self‐stability of polymer particles during the polymerization in the absence of surfactants was studied. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3098–3107, 2008     

聚L-丙氨酸-聚乙二醇嵌段共聚物的胶束化行为研究

以氨基聚乙二醇单甲醚(MPEG-NH₂)为大分子引发剂, 采用开环聚合方法合成了聚L-丙氨酸-聚乙二醇嵌段共聚物(PAME), 并对其结构进行了表征; 用圆二色谱(CD)研究了嵌段共聚物在水溶液中的二级结构, 用芘荧光探针技术研究了共聚物胶束的形成及其临界胶束浓度(CMC), 利用动态光散射(DLS)和透射电镜(TEM)研究了胶束的粒径分布和形态. 结果表明, 在水溶液中共聚物链以α-螺旋构象形式存在, 在一定条件下嵌段共聚物能够形成球形的稳定胶束, PAME-1形成胶束的CMC为1.99×10^-5 mol/L, CMC值受共聚物中聚L-丙氨酸(PLA)链段含量的影响.     

The pH induced vesicle to micelle morphology transition of a THP‐protected polymer

A diblock copolymer consisting of tetrahydropyranyl acrylate (THPA) as a pH‐deprotectable block, and a permanently hydrophobic block, methyl acrylate, was synthesized by RAFT polymerization using a quaternary amine functionalized, hydrophilic, RAFT chain transfer agent. The polymer self‐assembled in water to form vesicles with D_h = 130 nm, as determined by DLS and cryogenic transmission electron microscopy. Acid catalyzed deprotection of the THPA units to yield acrylic acid resulted in a vesicle to micelle morphology transition, as evidenced by the decrease in hydrodynamic diameter to D_h = 19 nm and the observation of micelles by dry state transmission electron microscopy. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 3026–3031