Nanomaterials with enzyme-like characteristics (nanozymes)_ next-generation artificial enzymes

纳米材料基本词汇Nanovector ?nanoswimmerNanosyringeNanopinNanodropletNanomedicine 纳米医学/纳米药物nanocube 纳米立方体纳米尺度 nanoscale纳米基元 nano-unit纳米结构单元 nanostructure unit纳米材料 nanomaterial纳米技术 nanotechnology纳米结构体系 nanostructure system纳米组装体系 nanostructure assembling system 纳米器件 nanodevice碳纳米管 carbon nanotubes，CNTs原子团簇 atom cluster单分散颗粒[系] monodispersed particle纳米颗粒 nanoparticle团粒 aggregate纳米粉体 nano-powder纳米纤维 nano-fibre/Nanofiber纳米薄膜 nano-film纳米块体 nano-bulk纳米孔 nano-pore纳米晶体材料 nanocrystalline material纳米非晶材料 amorphous nanomaterial纳米准晶材料 quasi-crystal nanomaterial金属纳米材料 metallic nanomaterial无机非金属纳米材料 inorganic non-metallic nanomaterial高分子纳米材料 polymer nanomaterial纳米复合材料 nanocomposites结构纳米材料 structured nanomaterial功能纳米材料 functional nanomaterial生物医用纳米材料 biomedical nanomaterial小尺寸效应 small-size effect表面效应 surface effect量子尺寸效应 quantum size effect宏观量子隧道效应 macroscopic quantum tunneling effect，MQT 惰性气体沉积法 inert gas deposition物理粉碎法 physics grinding高能球磨法 high energy ball mill溅射法 sputtering物理粉碎法 physics grinding爆炸法 explosion喷雾法 spraying冷冻干燥法 freeze drying化学气相沉积法 chemical vapor deposition，CVD沉淀法 precipitation水热合成法 hydrothermal synthesis溶胶-凝胶法 sol－gel辐射化学合成法 radiation chemical synthesis快速凝固法 rapidly quenching强烈塑性变形法 severe(intense) plastic deformation 非晶晶化法 amorphous solid crystallization溅射法 sputtering非晶晶化法 crystallization of amorphous solid原位复合法 in-situ composite插层复合法 intercalation hybrids微乳液法 micro emulsion模板合成法 template synthesis自组装法 self-assembly石墨电弧放电法 graphite arc discharge快速凝固法 rapidly quenching表面处理 surface treatment表面修饰 surface decoration稳定化处理 passivating treatmentX射线衍射法 X-ray diffractometry ，XRD扫描探针显微镜 scanning probe microscopy, SPM扫描隧道显微镜 scanning tunneling microscopy, STM扫描近场光学显微镜 scanning near-field optical microscopy, SNOM 原子力显微镜 atomic force microscopy, AFM扫描电容显微镜 scanning capacitance microscopy, SCM磁力显微镜 magnetic force microscopy, MFM扫描热显微镜 scanning thermal microscopy, STHMX射线衍射法 X-ray diffractometry ，XRDX射线衍射线宽化法 X-ray diffractometry line broadening, XRD-LB X射线小角度散射法 small angle X-ray scattering, SAXS透射电子显微镜法 transmission electron microscopy ,TEM透射电镜法 TEM method扫描电子显微镜法 scanning electron microscopy , SEM扫描电镜法 SEM method拉曼光谱法 raman spectrometry红外吸收光谱法 infrared absorption spectroscopy穆斯堡尔谱法 mossbauer spectrometry光子相关谱法 photon correlation spectroscopyBET法 BET压汞仪法 mercury porosimetry纳米压痕仪 nano impress,NI4.6.16扫描探针显微法 scanning probe microscopy, SPM扫描隧道电子显微法 scanning tunneling electron microscopy,STM 扫描近场光学显微法 scanning near-field optical microscopy，SNOM 原子力显微法 atomic force microscopy，AFM扫描电容显微法 scanning capacitance microscopy, SCM扫描热显微法 scanning thermal microscopy, STHM场离子显微法 field ion microscopy, FIM磁力显微法 magnetic force microscopy, MFM激光干涉仪 laser interferometer激光衍射/散射法 laser diffraction and scattering离心沉降法 centrifugal sedimentation。

序号期刊全称缩写1Nature Reviews Materials NAT REV MATER2NATURE MATERIALS NAT MATER3Nature Nanotechnology NAT NANOTECHNOL4Materials Today MATER TODAY5MATERIALS SCIENCE & ENGINEERING R-REPORTSMAT SCI ENG R6PROGRESS IN MATERIALS SCIENCE PROG MATER SCI7ADVANCED MATERIALS ADV MATER8Advanced Energy Materials ADV ENERGY MATER9Annual Review of Materials Research ANNU REV MATER RES 10ADVANCED FUNCTIONAL MATERIALS ADV FUNCT MATER11Materials Horizons MATER HORIZ12Nano Energy NANO ENERGY13INTERNATIONAL MATERIALS REVIEWS INT MATER REV14ACS Energy Letters ACS ENERGY LETT15Journal of Materials Chemistry A J MATER CHEM A16CHEMISTRY OF MATERIALS CHEM MATER17Small SMALL18npj Computational Materials NPJ COMPUT MATER19BIOMATERIALS BIOMATERIALS20ACS Applied Materials & Interfaces ACS APPL MATER INTER 21Advanced Optical Materials ADV OPT MATER22Nano-Micro Letters NANO-MICRO LETT23NPG Asia Materials NPG ASIA MATER242D Materials2D MATER25JOURNAL OF POWER SOURCES J POWER SOURCES26Biofabrication BIOFABRICATION27CURRENT OPINION IN SOLID STATE &MATERIALS SCIENCECURR OPIN SOLID ST M28Acta Biomaterialia ACTA BIOMATER29Materials Research Letters MATER RES LETT30ACTA MATERIALIA ACTA MATER31Journal of Materials Chemistry C J MATER CHEM C32Biomaterials Science BIOMATER SCI-UK33Advanced Healthcare Materials ADV HEALTHC MATER 34Advanced Electronic Materials ADV ELECTRON MATER 35CEMENT AND CONCRETE RESEARCH CEMENT CONCRETE RES 36COMPOSITES SCIENCE AND TECHNOLOGY COMPOS SCI TECHNOL37Materials Science & Engineering C-Materials for Biological ApplicationsMAT SCI ENG C-MATER38SOLAR ENERGY MATERIALS AND SOLAR CELLS SOL ENERG MAT SOL C 39COMPOSITES PART B-ENGINEERING COMPOS PART B-ENG40CORROSION SCIENCE CORROS SCI41Advanced Materials Interfaces ADV MATER INTERFACES 42MRS BULLETIN MRS BULL43SCIENCE AND TECHNOLOGY OF ADVANCEDMATERIALSSCI TECHNOL ADV MAT44Journal of Materials Chemistry B J MATER CHEM B45CEMENT & CONCRETE COMPOSITES CEMENT CONCRETE COMP 46Advanced Materials Technologies ADV MATER TECHNOL-US47MATERIALS & 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METALLURGIST+336AATCC Journal of Research AATCC J RES336Materia-Rio de Janeiro MATERIA-BRAZIL338AATCC REVIEW AATCC REV339JOURNAL OF CERAMIC PROCESSING RESEARCH J CERAM PROCESS RES 340POWDER METALLURGY AND METAL CERAMICS POWDER METALL MET C+341JOURNAL OF THE JAPAN INSTITUTE OFMETALSJ JPN I MET342Drewno DREWNO343RARE METAL MATERIALS AND ENGINEERING RARE METAL MAT ENG344TETSU TO HAGANE-JOURNAL OF THE IRONAND STEEL INSTITUTE OF JAPANTETSU TO HAGANE345Glass Technology-European Journal ofGlass Science and Technology Part AGLASS TECHNOL-PART A346Tekstil ve Konfeksiyon TEKST KONFEKSIYON 347Emerging Materials Research EMERG MATER RES348Soldagem & Inspecao SOLDAGEM INSP349JOURNAL OF NEW MATERIALS FORELECTROCHEMICAL SYSTEMSJ NEW MAT ELECTR SYS350APPITA APPITA351INTERNATIONAL JOURNAL OF POWDERMETALLURGYINT J POWDER METALL352MATERIALS EVALUATION MATER EVAL353ADVANCED MATERIALS & PROCESSES ADV MATER PROCESS 354MATERIALS PERFORMANCE MATER PERFORMANCE 355SEN-I GAKKAISHI SEN-I GAKKAISHI356PULP & PAPER-CANADA PULP PAP-CANADA357ZKG INTERNATIONAL ZKG INT358MOKUZAI GAKKAISHI MOKUZAI GAKKAISHI 359JCT COATINGSTECH JCT COATINGSTECH 360WOCHENBLATT FUR PAPIERFABRIKATION WOCHENBL PAPIERFABR 361SURFACE COATINGS INTERNATIONAL SURF COAT INTISSN影响因子2058-843751.941 1476-112239.235 1748-338737.49 1369-702124.5370927-796X24.480079-642523.75 0935-964821.95 1614-683221.875 1531-733115.846 1616-301X13.325 2051-634713.183 2211-285513.12 0950-660812.703 2380-819512.277 2050-74889.931 0897-47569.89 1613-68109.598 2057-39608.941 0142-96128.806 1944-82448.097 2195-10717.43 2311-67067.381 1884-40497.208 2053-15837.042 0378-7753 6.945 1758-5082 6.838 1359-0286 6.5481742-7061 6.383 2166-3831 6.161 1359-6454 6.036 2050-7526 5.976 2047-4830 5.831 2192-2640 5.609 2199-160X 5.466 0008-8846 5.43 0266-3538 5.16 0928-4931 5.080927-0248 5.018 1359-8368 4.92 0010-938X 4.862 2196-7350 4.834 0883-7694 4.788 1468-6996 4.7872050-750X 4.776 0958-9465 4.66 2365-709X 4.6220264-1275 4.525 1359-835X 4.514 0169-4332 4.439 2373-9878 4.432 0934-0866 4.384 2095-8226 4.318 1359-6462 4.163 2166-532X 4.127 0263-8223 4.101 2168-0396 4.091 0969-0239 3.809 0955-2219 3.794 0925-8388 3.779 1744-683X 3.709 1473-2262 3.667 1387-1811 3.649 0924-0136 3.6471005-0302 3.609 2079-4991 3.504 0950-0618 3.485 0966-9795 3.420921-5093 3.4140957-4484 3.404 2238-7854 3.398 1552-4973 3.373 0921-5107 3.316 0304-386X 3.3 1751-6161 3.239 1549-3296 3.231 2199-692X 3.173 0142-1123 3.132 0272-8842 3.057 2159-6859 3.008 0022-2461 2.993 1556-6560 2.993 2190-4286 2.968 0964-1726 2.9630043-1648 2.960002-7820 2.9560300-9440 2.955 1941-4900 2.917 0257-8972 2.906 1748-6041 2.897 1044-5803 2.892 1738-8090 2.882 0025-5408 2.873 1674-2001 2.785 0963-8695 2.7811099-6362 2.7760167-6636 2.6971438-7492 2.690167-577X 2.687 1042-6914 2.669 1537-6494 2.6450263-4368 2.606 1369-8001 2.593 1090-0268 2.592 1438-1656 2.576 1002-0071 2.572 2159-3930 2.566 8756-758X 2.533 0927-0256 2.53 1996-1944 2.467 1934-8630 2.455 0957-4530 2.448 1029-9599 2.38 2079-6412 2.35 2287-237X 2.333 0957-4522 2.324 0925-3467 2.32 1359-5997 2.271 0142-9418 2.247 0925-9635 2.232 0948-1907 2.227 1045-389X 2.211 0254-0584 2.21 1687-4110 2.2070737-0652 2.183 1606-5131 2.172 1047-4838 2.145 2073-4352 2.1440091-4037 2.1271388-0764 2.127 1431-9276 2.124 2051-672X 2.074 0042-207X 2.067 0143-7496 2.065 0267-0844 1.978 1058-9759 1.957 1598-9623 1.952 1059-9630 1.949 0272-8397 1.943 0040-6090 1.939 1960-6206 1.936 0021-8464 1.936 1362-1718 1.9360364-5916 1.935 0010-9312 1.927 2041-1286 1.912 0920-5063 1.911 2191-9089 1.904 1569-1713 1.8961073-5623 1.887 1380-2224 1.858 1073-5615 1.834 0465-2746 1.803 0267-0836 1.803 1003-6326 1.795 0965-0393 1.793 0017-1557 1.767 0734-2101 1.7610928-0707 1.7451847-9804 1.73 2075-4701 1.704 0195-9298 1.69 0334-6005 1.66 1434-5021 1.658 2158-3226 1.653 0043-2296 1.652 0288-4534 1.638 1478-6435 1.6321945-9645 1.6190021-9983 1.613 2226-4108 1.605 1475-1305 1.605 0883-9115 1.598 2158-5849 1.5970946-7076 1.581 0934-9847 1.567 0361-5235 1.566 0030-770X 1.547 0040-5175 1.54 1738-8228 1.518 1001-0521 1.5 0884-2914 1.495 0024-9831 1.488 0015-2684 1.483 2095-025X 1.478 1872-2105 1.4750731-6844 1.471 1976-4251 1.432 1580-3139 1.424 1611-3683 1.424 0960-3409 1.4230277-3813 1.418 0018-3768 1.401 1450-5339 1.4 2280-8000 1.397 1687-8434 1.372 1385-2000 1.3641533-4880 1.354 1229-9197 1.353 0915-1559 1.35 2049-1220 1.344 1006-7191 1.3411059-9495 1.34 0929-189X 1.333 8756-0879 1.333 1947-2935 1.318 1071-1023 1.314 1573-4137 1.306 0734-9041 1.296 1528-0837 1.2831464-4207 1.2812050-6252 1.268 1674-4799 1.261 0947-5117 1.259 0889-325X 1.252 0025-5289 1.248 1876-990X 1.246 1385-3449 1.238 2287-5301 1.237 1066-7857 1.232 0308-0501 1.22 2296-8016 1.211 0043-2288 1.206 0301-9233 1.205 1930-2126 1.202 0040-5000 1.174 1007-8827 1.1711546-542X 1.165 1820-6131 1.152 2053-1591 1.151 1346-8014 1.134 1539-445X 1.132 1083-5601 1.131 0283-2631 1.131 1006-706X 1.126 0924-3046 1.1241516-1439 1.103 1743-6753 1.092 1793-6047 1.084 1544-0478 1.076 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0734-14150.5161475-74350.5120738-79890.5 1000-324X0.49 0191-56650.49 0369-94200.486 0914-49350.4820144-03220.476 1927-63110.475 0361-76100.473 1425-81290.468 0967-39110.461 0003-55990.46 1392-13200.45 1067-82120.446 1222-53470.438 0334-64550.433 0026-08430.432 0963-69350.422 0034-85700.412 0091-10620.4 0026-06730.397 1454-41640.39 1068-820X0.387 1842-65730.3860032-678X0.384 0971-04260.366 1672-64210.36 0026-08940.347 2330-55170.34 1517-70760.34 1532-88130.333 1229-91620.327 1068-13020.326 0021-48760.312 1644-39850.311 1002-185X0.29 0021-15750.2841753-35460.282 1300-33560.266 2046-01470.2540104-92240.244 1480-24220.243 1038-68070.186 0888-74620.182 0025-53270.162 0882-79580.147 0094-14920.144 0037-98750.109 0316-40040.098 0949-02050.091 0021-47950.057 1547-00830.035 0043-71310.005 1754-09250。

MAC4LDFpis Rev 04/221Product InformationMetaPolyzyme, DNA freeSuitable for Microbiome researchMAC4LDFSynonym: Multilytic Enzyme Mix Storage Temperature –20 °CProduct DescriptionMetagenomics investigates all DNA that has been isolated directly from given single samples, such as environmental samples or biological organisms.1,2Metagenomics allows for the investigation of microbes that exist in extreme environments, and which have been historically difficult to isolate, culture, andstudy.3 Metagenomics has revealed the existence of novel microbial species.4 Applications ofmetagenomics work include public health dataanalysis,5,6 discovery of novel proteins, enzymes and natural products,7,8 environmental studies,9,10 and agricultural investigations.11,12Microbes are difficult to disrupt because the cell walls may form capsules or resistant spores. DNA can be extracted by using lysing enzymes such as lyticase, chitinase, zymolase, and gluculase to induce partial spheroplast formation. Spheroplasts are subsequently lysed to release DNA.MetaPolyzyme products (Cat. Nos. MAC4L, MAC4LDF) are based on a multi-lytic enzyme mixture, originally developed by Scott Tighe, for use in microbiome and DNA extraction efficiency studies, and formulated for effective lysis of microbiome samples from extreme environments. MetaPolyzyme was originally evaluated and developed in consultation and collaboration with the Association of Biomolecular Resource Facilities (ABRF) Metagenomics and Microbiome ResearchGroup (MMRG; formerly the Metagenomics Research Group, MGRG).13-16Studies of microbial communities have beenenhanced by the use of culture-independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. DNA contamination during sample preparation is a major problem of sequence-based approaches. Extraction reagents free of DNA contaminants are thus essential. MetaPolyzyme, DNA free was developed to address the need for DNA-free reagents, to minimize microbial DNA contamination from reagents. This productundergoes strict quality control testing to ensure the absence of detectable levels of contaminatingmicrobial DNA using 35 cycles PCR amplification of 16S and 18S rDNA using universal primer sets.Precautions and DisclaimerFor R&D use only. Not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.ReagentThe enzymes in MetaPolyzyme, DNA free are:• Mutanolysin • Achromopeptidase • Lyticase • Chitinase • Lysostaphin •LysozymeAll the enzymes are individually tested for theabsence of contaminating DNA using 16S and 18S PCR amplification.Mutanolysin (from Streptomyces globisporus )Mutanolysin is a muralytic enzyme (muramidase) that cleaves the β-N -acetylmuramyl-(1→4)-N -acetylglucosamine linkage of the bacterial cell wall peptidoglycan-polysaccharide, particularly the β(1→4) bond in MurNAc-GlcNAc.17 Mutanolysin particularly acts on many Gram-positive bacteria, where the enzyme’s carboxy -terminal moietiesparticipate in the recognition and binding of unique cell wall structures.MAC4LDFpis Rev 04/22AchromopeptidaseAchromopeptidase (known also as β-lytic protease 18) has potent bacteriolytic activity on many Gram-positive aerobic bacteria 19 with high lytic activity, against bacterial strains with the A1α chemotype (such as Aerococcus viridans ), and the A3αchemotype (such as Staphylococcus epidermidis ) for cell wall peptidoglycan structures. The enzyme has been reported to have particular recognition for Gly-X sites in peptide sequences, and for Gly-Gly and ᴅ-Ala-X sites in peptidoglycans.20Lyticase (from Arthrobacter luteus )Lyticase is useful in digestion of linear glucosepolymers with β(1→3) linkages, of yeast glycan coats and for spheroplast formation, and of the cell wall of active yeast cells.Chitinase (from Streptomyces griseus )Chitinase degrades chitin by enzymatic hydrolysis to N-acetyl-D-glucosamine. Degradation occurs via two consecutive enzyme reactions: •Chitodextrinase-chitinase, apoly(1,4-β-[2-acetamido-2-deoxy-D-glucoside])-glycanohydrolase, removes chitobiose units from chitin.•N-acetylglucosaminidase-chitobiase cleaves the disaccharide to its monomer subunits, N-acetyl-D-glucosamine (NAGA).Lysostaphin (from Staphylococcus staphylolyticus )Lysostaphin is a lytic enzyme with activity against Staphylococcus species, including S. aureus . Lysostaphin has hexosaminidase, amidase, and endopeptidase activities. It cleaves polyglycine crosslinks in the cellular wall of Staphylococcus species, which leads to cell lysis.21,22Lysozyme (from chicken egg white)Lysozyme hydrolyzes β(1→4) linkages betweenN -acetylmuraminic acid and N -acetyl-D-glucosamine residues in peptidoglycan, and betweenN -acetyl-D-glucosamine residues in chitodextrin. Lysozyme lyses the peptidoglycan cell wall of Gram-positive bacteria.23Storage/StabilityThis product ships at cooler temperature conditions. Long-term storage at –20 °C is recommended. Reconstituted solutions of MetaPolyzyme, DNA free may be stored at –20 °C, but long-term solution stability has not been examined.Preparation InstructionsBecause of the great diversity of samples formetagenomics studies, it will be necessary for each researcher to work out particular conditions for optimal sample preparation and treatment. It is recommended to reconstitute MetaPolyzyme, DNA free in sterile PBS buffer, pH 7.5 (no EDTA, calcium or magnesium present in solution). The following is a sample procedure, to be scaled appropriately:1. Add 100 µL of sterile PBS (pH 7.5) to 1 vial ofMetaPolyzyme, DNA free.1.1. Resuspend by gentle agitation or pipetting. 1.2. Set solution aside at 2-8 °C until Step 7. 2. Thoroughly suspend sample in sterile PBS(pH 7.5). 3. Add 200 µL of sample in PBS to a 2 mLpolypropylene microcentrifuge tube. 4. Optional pellet wash:4.1. To sample tube, add 1 mL of PBS (pH 7.5). 4.2. Vortex, centrifuge and remove supernatant. 4.3. Repeat pellet wash two more times ifneeded. 5. Resuspend pelleted sample in 150 µL of PBS(pH 7.5). Vortex thoroughly.6. Optional: if solution will sit for more than 4 hours,sodium azide may be added to 0.02%. 7. Add 20 µL (*) of MetaPolyzyme, DNA free tosample solution. 8. Incubate at 35 °C for 2-24 hours.9. Continue with standard DNA extraction protocol. (*) The optimal volume and concentration of MetaPolyzyme, DNA free may vary in different experiments.References1. Gilbert, J.A., and Dupont, C.L., Ann. Rev. MarineSci., 3, 347-371 (2011). 2. Kang, H.S., and Brady, S.F., J. Am. Chem. Soc.,136(52), 18111-18119 (2014). 3. Ufarté, L. et al., Biotechnol. Adv., 33(8),1845-1854 (2015). 4. Davison, M. et al., Photosynth. Res., 126(1),135-146 (2015). 5. Afshinnekoo, E. et al., Cell Syst., 1(1), 72-87(2015).The life science business of Merck operatesas MilliporeSigma in the U.S. and Canada.Merck and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt, Germany or its affiliates.All other trademarks are the property of their respective owners. Detailed information on trademarks is available via publicly accessible resources.© 2022 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.MAC4LDFpis Rev 04/22 DK,DT,GCY,TJ,RBG,SBC,MAM36.The MetaSUB International Consortium,Microbiome, 4, 24 (2016). [Erratum inMicrobiome, 4, 45 (2016).]7.Trinidade, M. et al., Front. Microbiol., 6, 890(2015).8.Coughlan, L.M. et al., Front. Microbiol., 6, 672(2015).9.Palomo, A. et al., ISME J., 10(11), 2569-2581(2016).10.Pold, G. et al., Appl. Environ. Microbiol., 82(22),6518-6530 (2016).11.Mitra, N. et al., J. Gen. Virol., 97(8), 1771-1784(2016).12.Theuns, S. et al., Infect. Genet. Evol., 43,135-145 (2016).13.Baldwin, D.A. et al., "Life at the Extreme", ABRFMetagenomics Research Group Poster 2015,presented at the ABRF 2015 Conference, St.Louis, MO, USA, March 28-31, 2015.14.Baldwin, D.A. et al., "Implementing NewStandards in Metagenomics and the ExtremeMicrobiome Project", ABRF MetagenomicsResearch Group Poster 2016, presented at theABRF 2016 Conference, Fort Lauderdale, FL, USA, February 20-23, 2016.15.McIntyre, A. et al., "Life at the Extreme: TheABRF Metagenomics Research Group", ABRFMetagenomics Research Group Poster 2017,presented at the ABRF 2017 Conference, SanDiego, CA, March 25-28, 2017.16.Tighe, S. et al., J. Biomol. Tech., 28(1), 31-39(2017).17.Gründling, A., and Schneewind, O., J. Bacteriol.,188(7), 2463-2472 (2006).18.Li, S.L. et al., J. Bacteriol., 172(11), 6506-6511(1990).19.Ezaki, T., and Suzuki, S., J. Clin. Microbiol.,16(5), 844-846 (1982). 20.Li, S. et al., J. Biochem., 124(2), 332-339(1998).21.Browder, H.P. et al., Biochem. Biophys. Res.Commun., 19, 383-389 (1965).22.Robinson, J.M. et al., J. Bacteriol., 137(3),1158-1164 (1979).23.Vocaldo, D.J. et al., Nature, 412(6849), 835-838(2001).NoticeWe provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose. The information in this document is subject to change without notice and should not be construed as a commitment by the manufacturing or selling entity, or an affiliate. We assume no responsibility for any errors that may appear in this document.Technical AssistanceVisit the tech service page at/techservice.Standard WarrantyThe applicable warranty for the products listed in this publication may be found at /terms. Contact InformationFor the location of the office nearest you, go to /offices.。

第23卷第2期2024年3月杭州师范大学学报(自然科学版)J o u r n a l o f H a n g z h o uN o r m a l U n i v e r s i t y(N a t u r a l S c i e n c eE d i t i o n )V o l .23N o .2M a r .2024收稿日期:2023-05-25 修回日期:2023-06-07基金项目:浙江省自然科学基金项目(L Q 20B 060003);杭州师范大学科研启动经费项目(2018Q D L 040).通信作者:汪红娣(1989 ),女,讲师,博士,主要从事靶向纳米药物的设计和控缓释研究.E -m a i l :w a n g .h o n gd i @h z n u .e d u .c n D O I :10.19926/j.c n k i .i s s n .1674-232X.2023.05.251文献引用:朱曙霞,王美玲,许紫宁,等.基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究[J ].杭州师范大学学报(自然科学版),2024,23(2):113-123.Z HU S h u x i a ,WA N G M e i l i n g ,X UZ i n i n g ,e t a l .S t u d y o n c o l o n -t a r ge t e d o r a l n a n o m e d i c i n e b a s e d o nZ e i n -H Ac a r r i e r [J ].J o u r n a l o fH a n g z h o uN o r m a lU n i v e r s i t y(N a t u r a l S c i e n c eE d i t i o n ),2024,23(2):113-123.基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究朱曙霞,王美玲,许紫宁,庾远龙,潘建林,汪红娣(杭州师范大学材料与化学化工学院,浙江杭州311121)摘 要:设计基于玉米醇溶蛋白(Z e i n )-透明质酸(HA )的口服纳米载体用于结肠靶向的药物递送.使用超声透析法制备玉米醇溶蛋白-透明质酸-羟基喜树碱(H C P T )纳米药物(Z e i n -HA@H C P T ),利用渴望函数分析法优化纳米药物制备条件,考察纳米药物的微结构㊁药物释放行为㊁细胞毒性和靶向摄取能力.Z e i n -HA@H C P T具有95%以上的药物包封率,有优异的生物学稳定性㊁生物相容性和独特的抗胃酸分解特性,结肠环境下的药物累积释放显著提升.Z e i n -HA@H C P T 通过C D 44受体介导的内吞作用被结肠癌细胞靶向摄取,可提高药物抗肿瘤疗效.关键词:玉米醇溶蛋白;透明质酸;羟基喜树碱;结肠靶向中图分类号:T Q 46 文献标志码:A 文章编号:1674-232X (2024)02-0113-11结直肠癌是常见的消化道恶性肿瘤,具有早期症状不明显㊁死亡率高㊁易复发等特点[1-2].抗癌口服药物制剂由于不受时空限制,易被患者接受和使用,具有极大的市场潜力[3].目前,因溶解度低㊁渗透性差或口服生物利用度低等原因,上市的化疗药物的相关产品多为静脉注射给药药物,如羟基喜树碱[4]㊁盐酸阿霉素[5]㊁紫杉醇[6]㊁吉西他滨[7]等.相较于注射给药,口服给药需考虑避免胃肠道p H ㊁消化酶和黏膜等多重生理屏障对药物活性和吸收率的影响[8],这就要求口服药物应具有良好的稳定性.此外,难溶或低渗类药物的溶解度低且不易跨过小肠上皮细胞膜,其口服制剂的开发最具挑战[9].将药物活性成分与合适载体材料结合,制备药物递送系统(d r u g d e l i v e r y s y s t e m ,D D S )是解决口服给药难题的重要技术手段.D D S 中的药物通常以分子态或无定形态存在,使药物溶解度显著提高.同时,D D S 中载体材料的合理选用可实现药物的跨膜递送㊁主动靶向和控缓释,从而提高治疗效果和安全性.玉米醇溶蛋白(Z e i n)具有独特的自组装特性㊁良好的抗胃酸分解特性和生物相容性,被广泛用作药物载体材料.但由于较强的疏水特性和缺乏靶向性,限制了玉米醇溶蛋白在纳米药物精准递送领域的应用[10].透明质酸(h ya l u r o n i ca c i d ,H A )是一种天然多糖,具有良好的亲水性,可被肿瘤细胞中过表达的411杭州师范大学学报(自然科学版)2024年C D44受体特异性识别[11].本研究利用超声透析法制备了具有独特抗胃酸分解特性和主动靶向能力的Z e i n-H A基纳米载体.结合超声乳化和透析技术的超声透析法(图1A),可有效避免Z e i n-H A纳米颗粒的聚集,提高产品分散性,同时可有效控制过程参数(如温度㊁溶剂㊁载材浓度㊁超声条件),调控玉米醇溶蛋白的自组装,获得高产品收率的纳米颗粒.在此基础上,利用纳米载体与羟基喜树碱(H C P T)之间的亲疏水作用,采用一步法获得高效封装的玉米醇溶蛋白-透明质酸基纳米药物,探究Z e i n-H A@H C P T的理化性质(形貌㊁化学结构㊁载药能力)㊁模拟胃肠道环境药物释放能力(图1B)和生化性质(生物稳定性㊁细胞毒性㊁靶向摄取能力等).A:超声透析法制备Z e i n-HA基纳米颗粒;B:口服纳米颗粒的胃肠道环境,依次经过胃酸环境(p H=1.2)㊁小肠流体(p H=7.4)和结肠流体(p H=6.0).图1玉米醇溶蛋白-透明质酸基纳米药物制备及递送F i g.1P r e p a r a t i o na n dd e l i v e r y r o u t e o fZ e i n-H Ab a s e dn a n o m e d i c i n e1材料与方法1.1主要试剂和仪器玉米醇溶蛋白购自美国S i g m a-A l d r i c h公司,透明质酸购自西安百川生物科技有限公司,D M E M细胞培养基㊁青霉素-链霉素购自赛澳美细胞技术(北京)有限公司,胰蛋白酶购自浙江吉诺生物医药技术有限公司,胎牛血清购自依科赛生物科技(太仓)有限公司,其余试剂(分析纯)均购自国药集团化学试剂有限公司.S U P R A T M55扫描电子显微镜㊁L S M710激光共聚焦显微镜㊁37081正置荧光显微镜均购自德国Z e i s s公司,H i t a c h iH T-7700透射电子显微镜购自日本日立公司,Z e t a s i z e rN a n oS90马尔文粒度仪购自德国M a l v e r n公司,D8A D V A N C EX射线衍射仪购自德国B r u h k e r公司,N i c o l e t i S50傅里叶变换红外光谱仪购自美国T h e r m oF i s h e r S c i e n t i f i c公司,S C I E N T Z-ⅡD超声波细胞粉碎机购自宁波新芝生物科技股份有限公司,L a m b d a750紫外分光光度计购自美国P e r k i n E l m e r公司,i M a r k1681130酶标仪购自美国B i o-R a d公司.1.2玉米醇溶蛋白-透明质酸纳米颗粒的合成玉米醇溶蛋白-透明质酸纳米颗粒(Z e i n-H A)使用超声透析的方法进行制备.超声透析技术装置主要包括超声细胞粉碎机㊁透析袋㊁夹套烧杯㊁恒温槽和隔音箱,如图1A所示.将2m g的H A溶于3m L的超纯水中,超声使H A充分溶解.随后,在此溶液中加入7m L无水乙醇和10m g玉米醇溶蛋白,充分混合溶解.将上述溶液置于透析袋中,悬挂于超声波细胞粉碎机中,设定超声功率为150W,o n/o f f=2s/2s, 20ħ反应20m i n.通过梯度离心(3000r/m i n5m i n,8000r/m i n10m i n,3次)收集Z e i n-H A.将收集得到的Z e i n -H A 进行冷冻干燥,获得淡黄色样品.1.3 羟基喜树碱的包载利用羟基喜树碱(H C P T )与Z e i n -HA 之间的亲疏水作用,制备Z e i n -H A@H C P T.将2m g 的H A 溶于3m L 的超纯水中,加入7m L 无水乙醇㊁10m g 玉米醇溶蛋白和3m g H C P T.将上述溶液置于透析袋中,悬挂于超声波细胞粉碎机中,以150W 的功率,o n /o f f =2s /2s ,20ħ反应20m i n ,制备得到Z e i n -H A@H C P T.1.4 载药前后纳米颗粒的储存稳定性测试将新鲜制备的Z e i n -H A 和Z e i n -H A@H C P T 纳米颗粒分别置于p H7.4的磷酸盐缓冲溶液(P B S )中重悬,分别在第1天㊁第3天㊁第7天㊁第14天时取出部分样品,使用动态光散射仪(D L S )测定粒径㊁粒度分布指数(p o l y d i s p e r s i t yi n d e x ,P D I )和Z e t a 电位.1.5 纳米颗粒的形貌表征使用扫描电子显微镜(S E M )在15k V 的加速电压下,观察纳米颗粒的表面形貌结构和粒径大小.使用透射电子显微镜(T E M )在亮场模式㊁100k V 的加速电压下,观察纳米颗粒的内部形貌特征.1.6 载药前后纳米颗粒的粒径和Z e t a 电位表征将Z e i n -HA 和Z e i n -H A@H C P T 离心重悬后分散于超纯水中,使用D L S 测定载药前后纳米颗粒的粒径㊁P D I 和Z e t a 电位.1.7 纳米颗粒的化学结构与晶型结构分析使用傅里叶红外光谱仪(F T I R )分析不同纳米颗粒的化学结构,设置扫描范围为4000c m -1~500c m -1.使用X 射线衍射仪(X R D )分析不同样品的晶型结构,设定仪器参数:在40m A 和40k V 下产生C u -K α辐射,扫描速度为10ʎ/m i n ,扫描范围为5ʎ~80ʎ.1.8 Z e i n -H A @H C P T 的载药量和包封率表征图2 H C P T 的标准曲线F i g.2 S t a n d a r d c u r v e o fH C P T 在Z e i n -HA@H C P T 中加入二甲基亚砜(D M S O ),超声使药物溶出,离心后取上清液.使用紫外分光光度计测定384n m 处H C P T 的吸光度,根据图2的标准曲线计算上清液中H C P T 的质量浓度.分别计算纳米颗粒的包封率E 和载药量L ,计算公式为E =m (被封装的H C P T )m (总H C P T )ˑ100%,(1)L =m (被封装的H C P T )m (总纳米颗粒)ˑ100%.(2)1.9 Z e i n -H A @H C P T 的体外释放药物实验准确称取5m g Ze i n -HA@H C P T 分散于透析袋中,置于透析液为模拟胃液的烧杯中,透析2h .2h 后将透析袋取出,置于透析液为模拟小肠液的烧杯中,继续透析4h .最后置于透析液为模拟结肠液的烧杯中,透析18h .每隔一段时间,在透析液中取出2m L 液体,采用紫外分光光度计测定吸光度,同时补充2m L 相应的透析液.根据图2标准曲线,确定Z e i n -H A@H C P T 的体外累积药物释放百分比.1.10 细胞培养在37ħ㊁含5%C O 2的湿润条件培养基中培养小鼠结直肠癌细胞(C T 26,C D 44受体高度表达).条件培养基为含有10%胎牛血清和1%抗生素(100U /m L 青霉素和100μg/m L 链霉素)的D M E M 培养基.1.11 纳米颗粒的细胞毒性实验通过C C K -8比色法确定H C P T ㊁Z e i n -H A 和Z e i n -H A@H C P T 对C T 26细胞生长的影响.将C T 26细胞接种在96孔板中(每孔5ˑ103个细胞),给予条件培养基贴壁培养24h .用不同质量浓度(H C P T 等511 第2期朱曙霞,等:基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究同质量浓度0㊁0.001㊁0.01㊁0.125㊁0.25㊁0.50㊁1㊁2.5㊁5㊁10μg/m L )的上述纳米颗粒分别处理细胞.培养24h 后,加入10μLC C K -8,继续在37ħ㊁5%C O 2的潮湿环境中孵育1h .用酶标仪读取450n m 下的吸光度值.1.12 纳米颗粒的细胞摄取实验和H A 竞争实验将C T 26细胞接种在6孔板中(每孔1.5ˑ106个细胞),孵育24h .分别给予0.5μg /m L H C P T 或Z e i n -H A@H C P T (H C P T 等同质量浓度为0.5μg/m L )孵育4h .随后,用细胞核染料D A P I 染色,P B S 清洗后封片.使用正置荧光显微镜观察细胞摄取情况.将C T 26细胞与游离0.50m g /m L HA 预处理1h 后,更换新鲜培养基.加入0.5μg/m L H C P T 或Z e i n -H A@H C P T (H C P T 等同质量浓度为0.5μg/m L ),与细胞共同孵育4h .去除培养基后,用D A P I 染色,使用正置荧光显微镜观察竞争实验结果.2 结果与讨论2.1 不同因素对Z e i n -H A 粒径㊁P D I 和Z e t a 电位的影响使用正交试验设计O A 16(44)探索不同实验条件对Z e i n -H A 尺寸的影响,并优化超声透析制备Z e i n -H A 纳米颗粒的操作参数.如表1所示,实验设置了4个因素,即玉米醇溶蛋白质量浓度㊁H A 分子量㊁反应温度和超声功率.每个因素的水平范围基于预实验结果确定,通过超声透析制备的Z e i n -H A 的粒径范围为201.5~2299.3n m.表1 O A 16(44)的正交实验结果T a b .1 R e s u l t s o f t h eO A 16(44)m a t r i x o f o p e r a t i n g co n d i t i o n s 实验序号Z e i n质量浓度/(m g /m L )HA分子量/k D a 反应温度/ħ超声功率/WZ e t a 电位/m V粒径/n m 粒度分布指数渴望函数指数S E M 对应图序号11.010020100-21.0ʃ0.8201.5ʃ0.20.2040.903图3a 21.040030125-8.7ʃ1.2265.5ʃ0.10.1180.99631.010*******-24.8ʃ2.0243.8ʃ0.30.2340.862图3b 41.0150050175-14.2ʃ0.6373.6ʃ0.20.1770.926图3c52.510030150-55.4ʃ0.7405.5ʃ0.30.1460.61162.540020175-46.7ʃ1.3432.9ʃ0.30.2740.663图3d 72.5100050100-57.3ʃ0.8430.7ʃ0.20.2240.56582.5150040125-58.8ʃ0.1573.2ʃ0.10.2380.526图3e 95.010040175-49.5ʃ0.31120.3ʃ0.60.1980.566105.040050150-44.1ʃ0.7844.2ʃ0.40.1800.659图3f115.010*******-43.0ʃ0.61190.7ʃ0.30.4560.526图3g125.0150030100-46.7ʃ1.1573.5ʃ0.40.4380.589137.510050125-71.5ʃ1.41216.4ʃ0.40.9460147.540040100-49.7ʃ0.3734.2ʃ0.70.4490.542157.5100030175-39.5ʃ0.72299.3ʃ0.50.1850图3h 167.5150020150-41.5ʃ0.21219.0ʃ0.60.3150.576图3i最优条件1.040020150-36.4ʃ0.6344.9ʃ0.30.248不同实验组制备得到的典型Z e i n -H A 的扫描电子显微镜图见图3.当Z e i n 质量浓度为1.0m g/m L 时,能得到粒径较小但团聚交联明显的胶束状颗粒;当Z e i n 质量浓度为2.5m g /m L 时,Z e i n -HA 的交联情况得到一定改善;随着Z e i n 质量浓度进一步提高至5.0m g /m L 时,Z e i n -H A 表现为均一且分散性较好的球形形态;当Z e i n 质量浓度为7.5m g/m L 时,Z e i n -H A 的形貌虽呈球形形态,但粒径较大(2μm ).因此,通过调节操作参数,可获得均匀粒度和良好球形形貌的Z e i n -H A.611杭州师范大学学报(自然科学版)2024年a i 分别对应实验序号1,3,4,6,8,10,11,15和16.图3 Z e i n -H A 纳米颗粒的扫描电子显微镜图F i g .3 S E Mi m a g e s o fZ e i n -H An a n o pa r t i c l e s 采用单因素分析法和渴望函数分析法对正交实验结果进行分析,综合考虑对产品性能影响较大的各评价指标,获取最佳操作条件,如表2所示.根据渴望函数分析法结果,得到不同因素对纳米颗粒表型的重要性顺序为Z e i n 质量浓度>H A 分子量>超声功率>反应温度.根据k i 值,确定使用超声透析法制备Z e i n -H A 纳米颗粒的最佳参数水平:Z e i n 质量浓度为1.0m g/m L ㊁H A 分子量为400k D a ㊁反应温度为20ħ㊁超声功率为150W.使用最优参数条件制备的Z e i n -H A 纳米颗粒的粒径为344.9n m ,在正交表的粒径数据范围内.表2 不同评价指标的极差分析结果T a b .2 R a n g e a n a l ys i s r e s u l t s f o r e v a l u a t i o n i n d i c a t o r s 影响因素Z e i n 质量浓度HA 分子量反应温度超声功率 粒径 k 1271.1735.9761.0485.0 k 2460.6569.2886.0811.4 k 3932.21041.1667.9678.1k 41367.2684.8716.21056.5 R 4384.51887.7872.22286.2 Z e t a 电位 k 1-17.2-49.4-38.0-43.7 k 2-54.5-37.3-37.6-45.5 k 3-45.8-41.1-45.7-41.5 k 4-50.6-40.3-46.8-37.5 R149.448.436.732.1 粒度分布指数 k 10.1830.3740.3120.329 k 20.2210.2550.2210.440 k 30.3180.2750.2800.219711 第2期朱曙霞,等:基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究811杭州师范大学学报(自然科学版)2024年表2(续)影响因素Z e i n质量浓度HA分子量反应温度超声功率k40.4740.2920.3820.209R1.1630.4730.6420.924渴望函数指数k10.9220.5200.6670.650k20.5910.7150.5490.512k30.5850.4880.6240.677k40.2800.6540.5370.539R2.5690.9090.5180.660在最优条件下制备得到的Z e i n-H A的扫描电子显微镜图和动态光散射仪图如图4所示,结果表明通过超声透析法获得的Z e i n-H A纳米颗粒尺寸较小且分布较窄.a:扫描电子显微镜图;b:动态光散射仪图;D p50:中值粒径;P D I:粒径分布指数.图4使用最优条件制备的Z e i n-H A的扫描电子显微镜和动态光散射仪图F i g.4S E Mi m a g e a n dD L S r e s u l t s o fZ e i n-H A p r e p a r e du n d e r t h e o p t i m a l c o n d i t i o n2.2Z e i n-H A的药物负载与理化性质2.2.1Z e i n-H A@H C P T的形貌与化学结构使用超声透析法一步制备得到Z e i n-H A@H C P T.如图5所示,制备得到的Z e i n-H A@H C P T纳米药物为表面光滑的球形颗粒,粒径比Z e i n-H A增加了约30n m,平均粒径为383.5n m.a:扫描电子显微镜图;b:动态光散射仪图;D p50:中值粒径;P D I:粒径分布指数.图5Z e i n-H A@H C P T的扫描电子显微镜和动态光散射仪图F i g.5S E Mi m a g e a n dD L S r e s u l t s o fZ e i n-H A@H C P T使用傅里叶红外光谱仪研究了不同纳米颗粒的化学结构成分.如图6所示,H C P T在1740c m-1处表现出内酯环结构中酯键的红外特征峰[12].与Z e i n-HA的红外光谱相比,Z e i n-H A@H C P T具有1740c m-1处的H C P T特征峰,表明H C P T被成功包封在Z e i n-H A中.图6 H C P T ㊁Z e i n -H A @H C P T ㊁Z e i n -H A 和Z e i n 的傅里叶红外光谱仪图F i g .6 F T I Rs pe c t r a o fH C P T ,Z e i n -H A @H C P T ,Z e i n -H Aa n dZ e i n图7 H C P T ㊁Z e i n -H A @H C P T 和Z e i n -H A 的紫外分光光度计图F i g .7 U V -v i s s p e c t r a o fH C P T ,Z e i n -H A @H C P Ta n dZ e i n -H A 为进一步确保药物被封装在Z e i n -HA 中,对载药前后的纳米颗粒进行紫外分光光度计分析.如图7所示,游离H C P T 的紫外吸收峰为384n m ,Z e i n -H A@H C P T 中可以清晰地观察到该特征峰,而Z e i n -H A 中没有紫外特征吸收峰.实验结果表明,H C P T 被有效地包封在Z e i n -HA 中.2.2.2 Z e i n -H A@H C P T 的载药量和包封率H C P T 通过疏水作用被装载到Z e i n -HA 纳米颗粒中.使用D M S O 使纳米颗粒溶出并释放包封药物,测定溶出上清液中的H C P T 质量浓度,计算得到Z e i n -H A@H C P T 的包封率为95.85%,载药量为7.98%.结果表明,Z e i n -H A@H C P T 具有较高的包封率,可能是由于HA 的交联网状结构在一定程度上提高了纳米颗粒的包封率,或者H A 分子可以通过氢键作用将生物活性分子包封[13].图8 H C P T ㊁Z e i n -H A @H C P T ㊁Z e i n -H A 和Z e i n 的X 射线衍射图F i g.8 X R Dc u r v e s o fH C P T ,Z e i n -H A @H C P T ,Z e i n -H Aa n dZ e i n 2.3 不同纳米颗粒的晶型分析通过X 射线衍射仪表征,发现H C P T 原料药具有尖锐的晶型峰.当H C P T 原料药被封装在Z e i n -H A 中时,尖锐的晶型峰消失了,如图8所示,表明当封装在Z e i n -H A 中时,H C P T 以无定形的形式存在,这有助于提高H C P T 的溶解度和生物利用度[14].2.4 模拟胃肠道液的体外释药研究口服药物在胃肠道中的累积释放情况是评估药物疗效的重要指标.本研究根据药物口服后在各消化部位的停留时间,模拟观察了纳米药物在胃肠道消化液中911 第2期朱曙霞,等:基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究021杭州师范大学学报(自然科学版)2024年H C P T的释放情况.如图9所示,游离H C P T在前30分钟表现出明显的药物突释现象,随着时间的增加, H C P T的释放速率显著降低,这主要由于晶型态的H C P T原药在水溶液中的溶解度很低,H C P T原药快速达到了溶解平衡.Z e i n-H A@H C P T在胃液(S G F)中的释放量(小于10%)显著低于H C P T原药(约30%),这得益于Z e i n载体优异的抗胃酸分解特性.Z e i n-H A@H C P T在结肠液(S C F)环境下,表现出显著的药物释放(约60%),表明Z e i n-H A@H C P T具备优异的结肠靶向性,主要原因可能是Z e i n载体的控缓释作用和p H响应的Z e i n载体的溶胀行为.据文献[15-16]报道,结肠中的肠道微生物能有效降解Z e i n基载体,有助于目标药物在结肠病灶部位的靶向释放.图9H C P T和Z e i n-H A@H C P T在模拟胃肠道消化液中的体外释药曲线F i g.9I nv i t r od r u g r e l e a s e c u r v e o fH C P Ta n dZ e i n-H A@H C P T i n s i m u l a t e d g a s t r o i n t e s t i n a l f l u i d s分别取出部分在胃液(S G F)消化后㊁小肠液(S I F)消化后和结肠液(S C F)消化后的Z e i n-H A@ H C P T,使用扫描电子显微镜观察其表面形貌的变化.如图10所示,未经消化的Z e i n-H A@H C P T呈光滑的球形且粒径均一;经S G F消化后,其形貌和粒径未发生明显变化;经S I F消化后,Z e i n-H A@H C P T的表面形貌发生了轻微变化,其表面出现部分溶胀粘连,由立体球形转变为扁平球形;经S C F消化后,Z e i n-H A@H C P T失去完整的球形,呈现大片黏连,这可能是由于S C F使Z e i n-H A载体发生明显溶胀,从而释放H C P T,造成了Z e i n-H A@H C P T形态的改变.R a w:原始样品;S G F:模拟胃液流体;S I F:模拟小肠流体;S C F:模拟结肠流体.图10不同胃肠道液环境下Z e i n-H A@H C P T的扫描电子显微镜图F i g.10S E Mi m a g e s o fZ e i n-H A@H C P Ta f t e r t r e a t m e n tw i t hSG F,S I Fa n dS C F2.5 Z e i n -H A @H C P T 的生物稳定性生物稳定性是评估纳米颗粒在生物体内利用效率的关键因素.本研究对制备得到的Z e i n -H A@H C P T 进行了储存稳定性测试.将制备得到的Z e i n -H A@H C P T 置于p H 7.4的P B S 中,使用动态光散射仪测定粒径㊁粒度分布指数和Z e t a 电位在14d 内的变化.如表3所示,2周内,Z e i n -H A@H C P T 的粒径㊁粒度分布指数和Z e t a 电位均无显著变化,表明Z e i n -H A@H C P T 具有良好的生物稳定性.表3 P B S 缓冲液(pH 7.4)中Z e i n -H A @H C P T 的颗粒性能T a b .3 P a r t i c l e p r o p e r t i e s o fZ e i n -H A @H C P Ta f t e r d i f f e r e n t s t o r a g e t i m e s i nP B Sb u f f e r (p H 7.4)存储时间平均粒径/n m 粒度分布指数Z e t a 电位/m V 新鲜制备383.5ʃ1.40.274-36.9ʃ1.91d 390.1ʃ2.60.226-42.5ʃ2.73d 396.8ʃ2.90.248-36.0ʃ0.87d 400.4ʃ2.50.232-31.9ʃ1.514d395.3ʃ3.20.333-30.4ʃ0.32.6 纳米颗粒的体外细胞毒性实验选用小鼠结肠癌细胞(C T 26细胞,C D 44高表达)探究Z e i n -H A@H C P T 对结肠癌细胞生长的影响.如图11所示,Z e i n -H A 空白载体对C T 26细胞几乎没有细胞毒性;随着载体浓度的增加,细胞活性呈现一定程度的增加,这可能是由于Z e i n -H A 的降解产物有助于C T 26的增殖.Z e i n -H A@H C P T 显示出比游离H C P T 更低的I C 50值,说明Z e i n -H A@H C P T 的细胞毒性更强,这可能是由于肿瘤细胞对纳米颗粒的摄取能力高于游离药物,且包封在Z e i n -H A@H C P T 中的H C P T 呈无定形形态,溶解度相较于游离H C P T 有所提高,进而使H C P T 的生物利用度提高;同时Z e i n -H A@H C P T 优异的C D 44靶向能力使C D 44受体高表达的C T 26肿瘤细胞对Z e i n -H A@H C P T 的摄取增加,从而表现出更强的细胞毒性.图11 C T 26细胞毒性实验结果F i g .11 I nv i t r o c y t o t o x i c i t y ofH C P T ,Z e i n -H A @H C P Ta n dZ e i n -H At oC T 26c e l l s 2.7 纳米颗粒对肿瘤细胞的靶向摄取能力为探究C T 26细胞对Z e i n -H A@H C P T 的摄取能力,本研究使用正置荧光显微镜观察Z e i n -H A@H C P T 的细胞摄取情况.如图12所示,随着H C P T 和Z e i n -H A@H C P T 与细胞的孵育时间从1h 延长至4h ,细胞内的荧光强度也逐渐增强,这表明H C P T 和Z e i n -H A@H C P T 被细胞内化的过程是时间依赖性的摄取过程.4h 时,H C P T 与Z e i n -H A@H C P T 的摄取能力差异与细胞毒性的结果吻合.为探究Z e i n -H A@H C P T 的C D 44靶向能力,通过设置H A 竞争实验,对贴壁生长24h 的C T 26细胞使用游离H A (0.5m g /m L )预处理1h 后,再给予不同的药物共同孵育4h ,观察肿瘤细胞对药物摄取能力的变化.如图12所示,C T 26细胞与Z e i n -H A@H C P T 共孵育的实验组中,细胞内几乎没有绿色荧121 第2期朱曙霞,等:基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究221杭州师范大学学报(自然科学版)2024年光;缺少C D44靶向配体的游离药物H C P T,即使在H A预处理后,细胞内仍显示出强烈的绿色荧光,具有较高的摄取能力.实验结果表明,Z e i n-H A@H C P T纳米颗粒是通过C D44受体介导的通路进行细胞摄取的.A:H C P T处理组;B:Z e i n-HA@H C P T处理组;标尺:40μm.图12C T26细胞中纳米颗粒的摄取能力F i g.12U p t a k e a b i l i t y o f n a n o p a r t i c l e s i nC T26c e l l s3结论抗结肠癌口服药物克服肠胃道p H㊁消化酶和黏膜等多重生理屏障,实现结肠病灶部位的靶向蓄积是当前面临的重要挑战.本研究使用超声透析法构建基于玉米醇溶蛋白-透明质酸的纳米复合载体,利用四因素四水平的正交实验优化实验参数,使用渴望函数法评价操作参数对多指标的影响.获取制备纳米颗粒的最优条件:Z e i n质量浓度为1.0m g/m L,HA分子质量为400k D a,反应温度为20ħ,超声功率为150W.使用一步法制备Z e i n-H A@H C P T纳米药物(包封率达95.85%),具备优异的抗胃酸分解特性,利用时间依赖性药物控制释放机制实现结肠靶向递送.通过考察Z e i n-H A@H C P T的生物稳定性㊁细胞毒性㊁细胞摄取等生物学性能,表明Z e i n-H A@H C P T纳米药物具有良好的生物稳定性㊁高效的细胞毒性和C D44受体靶向能力,可实现对结肠癌的靶向治疗.参考文献:[1]R E A C T TC O L L A B O R A T I V E ,Z A B O R OW S K IA M ,A B D I L EA ,e t a l .C h a r a c t e r i s t i c s o f e a r 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a m i c t h e r a p y o f g l i o b l a s t o m am e d i a t e d b y p l a t e l e t sw i t h u l t r a s o u n d -t r i g g e r e d d r u g r e l e a s e [J ].D r u g De l i v ,2023,30(1):2219429.[6]B R U C ESF ,C H O K ,N O I A H ,e t a l .G A S 6-A X L i n h i b i t i o nb y A V B -500o v e r c o m e s r e s i s t a n c e t o p a c l i t a x e l i ne n d o m e t r i a l c a n c e r b y d e c r e a s i n g t u m o r c e l l g l y c o l y s i s [J ].M o l C a n c e rT h e r ,2022,21(8):1348-1359.[7]K AM I S AWA T ,WO O DLD ,I T O IT ,e t a l .P a n c r e a t i c c a n c e r [J ].L a n c e t ,2016,388(10039):73-85.[8]S O S N I K A ,A U G U S T I N ER.C h a l l e n g e s i n o r a l d r u g d e l i v e r y o f a n t i r e t r o v i r a l s a n d t h e i n n o v a t i v e s t r a t e g i e s t o o v e r c o m e t h e m [J ].A d v D r u g De l i vR e v ,2016,103:105-120.[9]MA C E WA NSR ,C H I L K O T IA.F r o mc o m p o s i t i o nt oc u r e :as y s t e m se n g i n e e r i n g a p p r o a c ht oa n t i c a n c e rd r u g c a r r i e r s [J ].A n g e w C h e mI n tE dE n gl ,2017,56(24):6712-6733.[10]G I T E R U S G ,A L I M A ,O E YI .R e c e n t p r o g r e s si nu n d e r s t a n d i n g f u n d a m e n t a l i n t e r a c t i o n sa n da p p l i c a t i o n so fz e i n [J ].F o o d H yd r o c o l l ,2021,120:106948.[11]L E IC ,L I U XR ,C H E N QB ,e t a l .H y a l u r o n i c a c i d a n da l b u m i nb a s e dn a n o p a r t i c l e sf o r d r ug d e l i v e r y [J ].JC o n t r o lR e l e a s e ,2021,331:416-433.[12]J I N X ,A S G H A RS ,Z HU X T ,e t a l .I nv i t r o a n d i nv i v o e v a l u a t i o no f 10-h y d r o x y c a m p t o t h e c i n -l o a d e d p o l y (n -b u t y l c y a n o a c r y l a t e )n a n o p a r t i c l e s p r e p a r e db y m i n i e m u l s i o n p o l y m e r i z a t i o n [J ].C o l l o i d sS u r f BB i o i n t e r f a c e s ,2018,162:25-34.[13]C H E N S ,M C C L E M E N T S D J ,J I A N L ,e ta l .C o r e -s h e l lb i o p o l y m e rn a n o p a r t i c l e sf o rc o -d e l i v e r y o fc u r c u m i na n d p i pe r i n e :s e q u e n t i a l e l e c t r o s t a t i c d e p o s i t i o nof h y a l u r o n i c a c i d a n d c h i t o s a n s h e l l s o n t h e z e i n c o r e [J ].A C SA p p lM a t e r I n t e r f a c e s ,2019,11(41):38103-38115.[14]T OMA RD ,S I N G H PK ,H O Q U ES ,e t a l .A m o r p h o u s s y s t e m s f o r d e l i v e r y o f n u t r a c e u t i c a l s :c h a l l e n g e s o p p o r t u n i t i e s [J ].C r i tR e v F o o dS c iN u t r ,2022,62(5):1204-1221.[15]S I N HA V R ,K UM R I A R.M i c r o b i a l l y t r i g g e r e dd r u g d e l i v e r y to t h e c o l o n [J ].E u r JP h a r mS c i ,2003,18(1):3-18.[16]P A T E L M ,AM I N A.R e c e n t t r e n d s i nm i c r o b i a l l y a n d /o r e n z y m a t i c a l l y d r i v e n c o l o n -s p e c i f i c d r u g d e l i v e r y s ys t e m s [J ].C r i tR e vT h e r D r u g C a r r i e r S y s t ,2011,28(6):489-552.S t u d y o nC o l o n -t a r ge t e dO r a lN a n o m e d i c i n eB a s e do nZ e i n -H AC a r r i e r Z HUS h u x i a ,WA N G M e i l i n g ,X UZ i n i n g ,Y U Y u a n l o n g ,P A NJ i a n l i n ,WA N G H o n gd i (C o l le g e o fM a t e r i a l ,C h e m i s t r y a n dC h e m i c a l E n g i n e e r i n g ,H a n g z h o uN o r m a lU n i v e r s i t y ,H a n gz h o u311121,C h i n a )A b s t r a c t :O r a l n a n o c a r r i e r s f o r c o l o n -t a r g e t e dm e d i c i n ed e l i v e r y b a s e do nZ e i n -h y a l u r o n i ca c i d (HA )w e r ed e s i gn e d i n t h i s s t u d y .T h e n a n o m e d i c i n e o f Z e i n -HA -h y d r o x y c a m p t o t h e c i n (Z e i n -HA@H C P T )w a s p r e p a r e db y t h e u l t r a s o u n d d i a l y s i s m e t h o d .T h e p r e p a r a t i o n c o n d i t i o n s o f t h en a n o m e d i c i n ew e r eo p t i m i z e db y d e s i r a b i l i t y f u n c t i o n .T h em i c r o s t r u c t u r e ,d r u gr e l e a s e b e h a v i o r ,c y t o t o x i c i t y a n d t a r g e t e d c e l l u l a r u p t a k e a b i l i t y o f t h e n a n o m e d i c i n ew e r e i n v e s t i g a t e d .T h e r e s u l t s s h o w e d t h a t Z e i n -HA@H C P T p o s s e s s e dah i g hd r u g e n c a p s u l a t i o ne f f i c i e n c y o v e r 95%.M e a n t i m e ,i t e x h i b i t e d e x c e l l e n t b i o l o g i c a l s t a b i l i t y ,b i o c o m p a t i b i l i t y ,a n du n i q u e g a s t r i ca c i dr e s i s t a n c e .T h ec u m u l a t i v er e l e a s eo fd r u g s i nt h ec o l o n i ce n v i r o n m e n t w a s s i g n i f i c a n t l y i m p r o v e d .F u r t h e r m o r e ,Z e i n -HA@H C P T w a s t a r g e t e da n d t a k e nu p b y c o l o nc a n c e r c e l l s t h r o u ghC D 44r e c e p t o rm e d i a t e de n d o c y t o s i s ,w h i c hc a n i m p r o v e t h e a n t i -t u m o r e f f i c a c y o f d r u gs .K e y wo r d s :Z e i n ;h y a l u r o n i c a c i d ;h y d r o x y c a m p t o t h e c i n ;c o l o n -t a r g e t e d 321 第2期朱曙霞,等:基于玉米醇溶蛋白-透明质酸载体的结肠靶向口服纳米药物研究。

2 ＤＯＩ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－５２５６．２０２３．０１．０２８细胞器之间相互作用在非酒精性脂肪性肝病发生发展中的作用刘天会首都医科大学附属北京友谊医院肝病中心，北京１０００５０通信作者：刘天会，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）摘要：细胞器除了具有各自特定的功能外，还可与其他细胞器相互作用完成重要的生理功能。

细胞器之间相互作用的异常与疾病的发生发展密切相关。

近年来，细胞器之间相互作用在非酒精性脂肪性肝病（ＮＡＦＬＤ）发生发展中的作用受到关注，特别是线粒体、脂滴与其他细胞器之间的相互作用。

关键词：非酒精性脂肪性肝病；细胞器；线粒体；脂肪滴基金项目：国家自然科学基金面上项目（８２０７０６１８）ＲｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅＬＩＵＴｉａｎｈｕｉ．（ＬｉｖｅｒＲｅｓｅａｒｃｈＣｅｎｔｅｒ，ＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌ，ＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｂｅｉｊｉｎｇ１０００５０，Ｃｈｉｎａ）Ｃｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ：ＬＩＵＴｉａｎｈｕｉ，ｌｉｕ＿ｔｉａｎｈｕｉ＠１６３．ｃｏｍ（ＯＲＣＩＤ：００００－０００１－６７８９－３０１６）Ａｂｓｔｒａｃｔ：Ｉｎａｄｄｉｔｉｏｎｔｏｉｔｓｏｗｎｓｐｅｃｉｆｉｃｆｕｎｃｔｉｏｎｓ，ａｎｏｒｇａｎｅｌｌｅｃａｎａｌｓｏｉｎｔｅｒａｃｔｗｉｔｈｏｔｈｅｒｏｒｇａｎｅｌｌｅｓｔｏｃｏｍｐｌｅｔｅｉｍｐｏｒｔａｎｔｐｈｙｓｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｓ．Ｔｈｅｄｉｓｏｒｄｅｒｓｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓａｒｅｃｌｏｓｅｌｙａｓｓｏｃｉａｔｅｄｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｖａｒｉｏｕｓｄｉｓｅａｓｅｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｔｈｅｒｏｌｅｏｆｏｒｇａｎｅｌｌｅｉｎｔｅｒａｃｔｉｏｎｓｈａｓａｔｔｒａｃｔｅｄｍｏｒｅａｔｔｅｎｔｉｏｎｉｎｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ，ｅｓｐｅｃｉａｌｌｙｔｈｅｉｎｔｅｒａｃｔｉｏｎｓｂｅｔｗｅｅｎｍｉｔｏｃｈｏｎｄｒｉａ，ｌｉｐｉｄｄｒｏｐｌｅｔｓ，ａｎｄｏｔｈｅｒｏｒｇａｎｅｌｌｅｓ．Ｋｅｙｗｏｒｄｓ：Ｎｏｎ－ａｌｃｏｈｏｌｉｃＦａｔｔｙＬｉｖｅｒＤｉｓｅａｓｅ；Ｏｒｇａｎｅｌｌｅｓ；Ｍｉｔｏｃｈｏｎｄｒｉａ；ＬｉｐｉｄＤｒｏｐｌｅｔｓＲｅｓｅａｒｃｈｆｕｎｄｉｎｇ：ＮａｔｉｏｎａｌＮａｔｕｒａｌＳｃｉｅｎｃｅＦｏｕｎｄａｔｉｏｎｏｆＣｈｉｎａ（８２０７０６１８） 细胞器可以通过膜接触位点与其他细胞器相互作用，完成物质与信息的交换，形成互作网络［１］。

化工进展Chemical Industry and Engineering Progress2022年第41卷第8期纳米纤维素构建超疏水材料研究进展詹洵，陈健，杨兆哲，吴国民，孔振武，沈葵忠（中国林业科学研究院林产化学工业研究所，江苏省生物质能源与材料重点实验室，国家林业和草原局林产化学工程重点实验室，林木生物质低碳高效利用国家工程研究中心，江苏省林业资源高效加工利用协同创新中心，江苏南京210042）摘要：纳米纤维素表面富含活性羟基，具有高度的亲水性和吸水性，这在很大程度上成为影响纳米纤维素在工业上大规模应用的主要因素。

对纳米纤维素表面的活性羟基进行化学修饰提高其疏水性，日益成为国内外学者研究的热点。

本文在简要阐述超疏水材料基本特征和制备方法的基础上，对比了不同超疏水材料制备方法（模板法、喷涂法、沉积法、刻蚀法）的优劣，重点介绍了国内外学者利用纳米纤维素构建超疏水材料（气凝胶、纸张、涂层、薄膜等）在生物医学、造纸工业、油水分离、食品包装、储能材料等不同领域的研究进展，归纳并分析了目前纳米纤维素构建超疏水材料在改性方式和性能提升等方面仍存在的问题，同时指出了纳米纤维素构建超疏水材料未来将朝着过程无污染化、工艺简化、稳定性优化等方向发展。

关键词：纳米纤维素；超疏水材料；气凝胶；涂层；薄膜中图分类号：TQ35文献标志码：A文章编号：1000-6613（2022）08-4303-11Progress on superhydrophobic materials from nanocelluloseZHAN Xun ，CHEN Jian ，YANG Zhaozhe ，WU Guomin ，KONG Zhenwu ，SHEN Kuizhong(Institute of Chemical Industry of Forest Products,Chinese Academy of Forestry;Key Laboratory of Biomass Energy andMaterial,Jiangsu Province;Key Laboratory of Chemical Engineering of Forest Products,National Forestry and Grassland Administration;National Engineering Research Center of Low-Carbon Processing and Utilization of Forest Biomass;Jiangsu Co -Innovation Center of Efficient Processing and Utilization of Forest Resources,Nanjing 210042,Jiangsu,China)Abstract:Due to the abundant hydroxyl groups on the surface,nanocellulose has high hydrophilicity and water absorption,which has become the main factor affecting its large-scale application.Functional modification of the active hydroxyl groups on the surface of nanocellulose to improve its hydrophobicity has increasingly become an attractive research area.Based on a brief description of superhydrophobic materials and a comparison of different preparation methods of superhydrophobic materials,this article focused on the research progress on using nanocellulose to construct superhydrophobic materials (aerogels,paper,coatings and films)in the fields of biomedical,papermaking,oil-water separation,food packaging,energy storage materials,etc .,and summarized and analyzed the problems in the application of nanocellulose superhydrophobic materials.At the same time,it was pointed out that the future development direction of nanocellulose to construct superhydrophobic materials would focus on the pollution-free process,process simplification and stability optimization.综述与专论DOI ：10.16085/j.issn.1000-6613.2021-2005收稿日期：2021-09-23；修改稿日期：2021-12-03。

DOI:10.1021/la904014z 6083Langmuir 2010,26(9),6083–6085Published on Web 03/18//Langmuir ©2010American Chemical SocietyGraphene Oxide as a Matrix for Enzyme ImmobilizationJiali Zhang,†,§Feng Zhang,‡,§Haijun Yang,†Xuelei Huang,‡Hui Liu,‡Jingyan Zhang,*,‡andShouwu Guo*,††National Key Laboratory of Micro/Nano Fabrication Technology,Research Institute of Micro/Nano Science and Technology,Shanghai Jiao Tong University,Shanghai,200240China,and ‡State Key Laboratory of Bioreactor Engineering,School of Pharmacy,East China University of Science &Technology,Shanghai,200237China.§These authors contributed equally to this work.Received October 16,2009.Revised Manuscript Received January 25,2010Graphene oxide (GO),having a large specific surface area and abundant functional groups,provides an ideal substrate for study enzyme immobilization.We demonstrated that the enzyme immobilization on the GO sheets could take place readily without using any cross-linking reagents and additional surface modification.The atomically flat surface enabled us to observe the immobilized enzyme in the native state directly using atomic force microscopy (AFM).Combining the AFM imaging results of the immobilized enzyme molecules and their catalytic activity,we illustrated that the conformation of the immobilized enzyme is mainly determined by interactions of enzyme molecules with the functional groups of GO.IntroductionGraphene oxide (GO),as a basic material for the preparation of individual graphene sheets in bulk-quantity,has attracted great attention in recent years.1-3In addition,the incredibly large specific surface area (two accessible sides),the abundant oxygen-containing surface functionalities,such as epoxide,hydroxyl,and carboxylic groups,and the high water solubility afford GO sheets great promise for many more applications.1,2For instance,the GO nanosheets modified with polyethylene glycol have been employed as aqueous compatible carriers for water-insoluble drug delivery.4The intrinsic oxygen-containing functional groups were used as initial sites for deposition of metal nanoparticles and organic macromolecules,such as porphyrin,on the GO sheets,which opened up a novel route to multifunctional nanometer-scaled catalytic,magnetic,and optoelectronic materials.5-7How-ever,few studies about the binding of biomacromolecules,such as enzymes,to GO have been reported to date.Since the discovery of the advantageous property of immobi-lized enzymes,the challenges in this area have been to explore new substrate materials with appropriate structures (including the morphology and surface functionality)and compositions to deepen the understanding of enzyme immobilization and thus to improve the catalytic efficiency of the immobilizedenzymes.8-11Recently,along with the development of nano-structured materials,a range of nanomaterials with different sizes and shapes have been utilized as the substrates for enzyme immobilization.12-14It has been demonstrated that the enzymes immobilized on the nanostructured materials have some advan-tages over the bulk solid substrates.8,15However,similar to bulk solid substrates,to efficiently immobilize enzymes on nanostruc-tured material surfaces,in many cases,labored work was required to modify/functionalize the substrate surface.16,17Moreover,for most of the nanostructured materials,it is hard to fully char-acterize their surfaces using conventional surface analytical tools.This limits the deep understanding of enzyme immobilization.Consequently,new nanostructured materials that not only can immobilize the enzyme enthusiastically but also can enable insight into the interactions between enzymes and the substrate are still in need of exploration.GO sheets should be an ideal substrate for the study of enzyme immobilization on nanostructured materials.As aforementioned,the individual GO sheet is enriched with oxygen-containing groups,which makes it possible to immobilize enzymes without any surface modification or any coupling reagents.The atomi-cally flat surface of GO should provide a platform to characterize the immobilized enzyme using conventional surface imaging techniques,such as atomic force microscopy (AFM),and to further study the interactions between enzyme molecules and the GO surface.We describe herein the immobilization of horse-radish peroxidase (HRP)and lysozyme,as model enzymes,on the GO.The enzyme immobilization was characterized in situ with AFM in a liquid cell,and the catalytic activity of the immobilized HRP was assayed using phenol and hydrogen peroxide as catalytic reaction substrates.*To whom correspondence should be addressed.E-mail:swguo@ (S.G.);jyzhang@ (J.Z.).(1)Li,D.;Muller,M.B.;Gilje,S.;Kaner,R.B.;Wallance,G.G.Nature Nanotechnol.2008,3,101–105.(2)Park,S.;Ruoff,R.S.Nature Nanotechnol.2009,4,217–223.(3)Tung,V.C.;Allen,M.J.;Yang,Y.;Kaner,R.B.Nature Nanotechnol.2009,4,25–29.(4)Liu,Z.;Robinson,J.T.;Sun,X.;Dai,H.J.Am.Chem.Soc.2008,130,10876–10877.(5)Lomeda,J.R.;Doyle,C.D.;Kosynkin,D.V.;Hwang,W.;Tour,J.M.J.Am.Chem.Soc.2008,130,16201–16206.(6)Muszynski,R.;Seger,B.;Kamat,P.V.J.Phys.Chem.C 2008,112,5263–5266.(7)Xu,Y.;Liu,Z.;Zhang,X.;Wang,Y.;Tian,J.;Huang,Y.;Ma,Y.;Zhang,X.;Chen,Y.Adv.Mater.2009,21,1275–1278.(8)Bornscheuer,U.T.Angew.Chem.,Int.Ed.2003,42,3336–3337.(9)Betancor,L.;Luckarift,H.R.Trends Biotechnol.2008,26,566–572.(10)Badalo,A.;Gomez,J.L.;Gomez,E.;Bastida,J.;Maximo,M.F.Chemo-sphere 2006,63,626–632.(11)Chen,B.;Pernodet,N.;Rafailovich,M.H.;Bakhtina,A.;Gross,ngmuir 2008,24,13457–13464.(12)Kim,J.;Grate,J.W.;Wang,P.Chem.Eng.Sci.2006,61,1017–1026.(13)Zhi,C.;Bando,Y.;Tang,C.;Golberg,D.J.Am.Chem.Soc.2005,127,17144–17145.(14)Tsang,S.C.;Yu,C.H.;Gao,X.;Tam,K.J.Phys.Chem.B 2006,110,16914–16922.(15)Takahashi,H.;Li,B.;Sasaki,T.;Miyazaki,C.;Kajino,T.;Inagaki,S.Chem.Mater.2000,12,3301–3305.(16)Lee,Y.M.;Kwon,O.Y.;Yoon,Y.J.;Ryu,K.Biotechnol.Lett.2006,28,39–43.(17)Lin,Y.;Lu,F.;Tu,Y.;Ren,Z.Nano Lett.2004,4,191–195.Letter Zhang et al.Experimental SectionGO was prepared using natural graphite powder through amodified Hummers method.18,19The as-obtained yellow-brownaqueous suspension of GO was stored at RT on a lab bench,and used for characterizations and enzyme immobilization.Thesamples for Fourier transform infrared(FT-IR)measurementwere prepared by grinding the dried powder of graphene oxidewith KBr together and then compressing the mixture into thinpellets(EQUINOX55,Bruker,Germany).The specimens oftransmission electron microscopy(TEM)(JEM-2010)were pre-pared by placing the aqueous suspension(∼0.02mg/mL)ofgraphene oxide on the carbon-coated copper grids,and blottedafter30s.AFM images of graphene oxide were taken on aMultiMode Nanoscope V scanning probe microscopy system(Veeco).The samples for AFM were prepared by dropping theaqueous suspension(∼0.02mg/mL)of GO on a freshly cleavedmica surface.AFM images of the GO-bound enzymes wereacquired in a liquid cell using tapping mode.To acquire in situAFM images for enzyme immobilization,the liquid cell wascirculated with the fresh enzyme solution during imaging.20Enzyme immobilization was carried out by adding the desiredamount of GO to0.1M phosphate buffer that contained theenzymes to be immobilized.21The mixture was incubated for30min on ice with shaking and then centrifuged.The supernatantwas used to determine the enzyme loading.The immobilized enzy-mes were washed three times with the same buffer to remove physi-cal adsorbed enzymes.The resulting immobilized enzymes werethen subjected to activity assay.A colorimetric assay was employedto evaluate HRP activity.22The initial reaction rates were obtainedvia a linear fit of the curve of the product absorbance at510nmversus the reaction time(Supporting Information Figure S2).23Results and DiscussionThe morphology of as-prepared GO was characterized firstusing AFM(Figure1a).The height of the flat GO sheet is∼1nm(Figure1b),demonstrating a single atomic layer thicknessstructure feature.The thin nanoplate motif of the GO sheetswas also confirmed by TEM(Figure1c).The functional groups (Figure1d)existing on the GO surface were verified by FT-IR spectroscopy(Supporting Information Figure S1).The enzyme immobilization was carried out by incubating the GO(0.5to 1mg/mL aqueous dispersions)with the enzymes in phosphate buffer solution at4°C.We found that HRP can be spontaneously immobilized on GO.Presumably,the amine groups of HRP may form amide bonds with the carboxylic groups of GO;however, without any coupling reagents,this covalent interaction usually happens very slowly.24Therefore,the covalent bonding may not contribute to HRP-GO interaction.To elucidate the contribu-tion of other interactions,the phosphate buffers with pH from4.8 to8.8,were tested.As shown in Figure2,the loading of HRP on the GO decreases with increasing pH.HRP(pI=7.2)has a net positive charge at pH below7.2and a net negative charge at pH above7.2.The GO sheets are negatively charged in the aqueous solution with a pH range from4to11(see Supporting Informa-tion Figure S3).1-3Thus,in the buffer solutions with a pH range from4.8to7.2,the positively charged HRP interacts with the negatively charged GO by electrostatic interaction,while in the buffer solutions from pH7.2to8.8,HRP and GO both are negatively charged,and will repel each other.Therefore,less HRP was loaded.Only an∼30%enzyme loading decrease was observed when the pH of the buffer solutions increased from 4.8to8.8(Figure2),suggesting that other interactions,such as hydrogen bonding between the oxygen-containing functionalities of GO and surface amino acid residues of HRP,may contribute to GO-HRP interaction,too.Owing to the strong electrostatic interactions and hydrogen bonding,the maximum loading of HRP on GO at pH7.0is about100μg/mg of GO,which is much higher than the loadings on many reported materials.25-27To further illustrate the electrostatic interaction between the enzymes and GO,we examined the immobilization of lysozyme,an enzyme with pI=10.3(positively charged at pH7.0).The lysozyme can be spontaneously immobilized on GO,too,with the maximum loading of about700μg/mg of GO at pH7.0.The positively charged surface of lysozyme apparently is favorable for its interaction with GO.The loading difference between HRP and lysozyme indicates that the interactions of substrate-enzymes are determined by the surface charges of the specified enzymes and the substrate.The high enzyme loadings reveal the exceptional potential of GO as a solid substrate for enzyme immobilization. The enzyme immobilization was monitored in situ using AFM. Figure3a and b shows typical AFM images of the GO in a liquid Figure1.(a)Tapping mode AFM image of graphene oxide(GO) on a mica surface,(b)height profile of the AFM image,(c)TEM image of the GO,and(d)schematic model of GO.Figure2.pH influence on HRP loading.Conditions:50μg GO and2μg/mL HRP.(18)Hummers,W.S.;Offerman,R.E.J.Am.Chem.Soc.1958,80,1339–1339.(19)He,H.;Klinowski,J.;Forster,M.;Lerf,A.Chem.Phys.Lett.1998,287,53–56.(20)Guo,S.;Ward,M.D.;Wesson,ngmuir2002,18,4284–4291.(21)Cheng,J.;Ming,Yu,S.;Zuo,P.Water Res.2006,40,283–290.(22)Nicell,J.A.;Wright,H.Enzyme Microb.Technol.1997,21,302–310.(23)Buchanan,I.D.;Nicell,J.A.Biotechnol.Bioeng.1997,54,251–261.(24)Cao,Y.;Kyratzis,I.Bioconjugate Chem.2008,19,1945–1950.(25)Pundir,C.S.;Malik,V.;Bhargava,A.K.;Thakur,M.;Kaliam,V.;Singh, S.;Kuchhal,N.K.J.Plant Biochem.Biotechnol.1999,8,123–126.(26)Azevedo,A.M.;Vojinovic,V.;Cabral,J.M.S.;Gibson,T.D.;Fonseca, L.P.J.Mol.Catal.B:Enzym.2004,28,121–128.(27)G o mez,J.L.;B o dalo,A.;G o mez,E.;Bastida,J.;Hidalgo,A.M.;G o mez, M.Enzyme Microb.Technol.2006,39,1016–1022.6084DOI:10.1021/la904014z Langmuir2010,26(9),6083–6085DOI:10.1021/la904014z6085Langmuir 2010,26(9),6083–6085Zhang et al.Lettercell after being incubated together with HRP in phosphate buffer for 30min.With a lower enzyme loading (HRP/GO =3:500,in weight),the particles (bright spots,presumably the immobilized enzyme molecules)on the GO surface were observed (Figure 3a).The average diameter and height of the particles on the GO surfaceare about 140and 15A,respectively.The dimension size of the immobilized HRP molecule,140Â140Â15A,is roughly con-sistent with the dimension size of free HRP,30Â65Â75A3.28This is the first picture of the native immobilized enzyme.The larger average diameter and shorter height of the immobilized HRP molecules revealed that immobilization induced some conforma-tional changes of the HRP molecules.With a higher enzyme loading (HRP/GO =3:50,in weight),the enzyme molecules tethered densely over all of the GO surface in the AFM image (Figure 3b).The distribution of HRP on the GO surface should be determined by the intrinsic sites of the oxygen functionalities.Except for the carboxylic groups,which are located at the periphery,others,such as hydroxyl and epoxide groups,distributed randomly over the GO surface.19The mole ratio of C/O of the GO used in the work is about 4,and thus,HRP may densely bind on the GO surface.This is in agreement with the AFM image (Figure 3b)where we observed the increased surface coverage with higher enzyme loading.The catalytic property of the HRP immobilized on GO was investigated using phenol as a reducing substrate.We found that the initial catalytic reaction rates of the immobilized HRP were linear to the HRP loading under an excess and constant substrate concentration (Figure 3d),though they are relatively lower than that of free HRP.29This result suggested that the voids presented between the immobilized HRP molecules are enough for the free diffusion of substrate and product into and out of the HRP active sites,though the immobilized enzymes seem crowded on the GO surface (see Figure 3b).Given the single atomic layer feature ofthe GO sheet,the total surface area is about 7.05Â1022A2/g,and assuming the average transverse area of one molecule HRP isabout 3000A2,the HRP molecules cover less than 50%of the surface area of GO even with the higher enzyme loading.The catalytic activities of the HRP immobilized on GO with the lower and higher enzyme loadings were further characterized by turnover number (K cat )and enzyme efficiency (K cat /K m ).K m and K cat values were obtained according to the Lineweaver -Burk equation as described in the Supporting Information (Figure S2).The values of the kinetic parameters K m and K cat are summarized in Table 1.The similar K m values for the GO immobilized HRP with the lower and higher enzyme loadings,and free HRP indicated that they all have a similar affinity to the reducing substrate.However,K cat /K m values of the immobilized HRP are lower than those of free HRP.Noticeably,the comparable K cat /K m values for the HRP immobilized on GO with the higher and lower enzyme loadings confirmed that increasing enzyme loading does not affect the enzyme efficiency.The catalytic reactions of the immobilized HRP (with the higher and lower enzyme loadings)with a bulky reducing substrate,2,4,6-trimethylphenol,exhibited similar activity,further supporting this result.Thus,combined with the AFM imaging results,we believe that the observed lower enzymatic activity for the immobilized HRP is mainly due to the HRP conformational changes induced by its binding to GO.According to the number of oxygen containing groups on the GO surface and the transverse area of one HRP molecule,there should be at least an average of two oxygen containing groups of the GO surface interacting with one HRP molecule (Figure 3c).Multiple interactions between the substrate and the enzyme molecule could change the enzyme conforma-tion.11Thus,to maintain the conformation and catalytic cap-ability of the immobilized enzyme,the distribution,number,and property of the functional groups on the substrate surface must be optimized to match the surface of the enzyme being immobilized.ConclusionIn summary,we have demonstrated that individual GO sheets could be used as substrates to study enzyme immobilization.Pronouncedly,the rich surface functional groups of GO make the immobilization of the enzymes happen quickly through electro-static interaction without using any cross-linking reagents;the unique flat surface of GO made it possible to observe the native immobilized enzyme in situ using AFM.We found that the catalytic performance of the immobilized enzymes is determined by the interaction of enzyme molecules with the surface functional groups of the substrate,but the enzyme specific activity is not influenced by the enzyme loading as far as the substrate surface was not fully covered by the enzyme.Based on the AFM images and enzyme activity assay,we conclude that full retention of the conformation of immobilized enzyme should be the key to improve its catalytic performance.Acknowledgment.This work was supported by the National “973Program”(Nos.2007CB936000and 2010CB933900)and the NSFC of China (Nos.20774029and 20671034).Supporting Information Available:FT-IR spectrum of GO and catalytic data of the immobilized HRP.This material is available free of charge via the Internet at .Figure 3.Tapping mode AFM images of the GO-bound HRPwith (a)lower and (b)higher enzyme loadings acquired in a liquid cell.(c)Schematic model of the GO-bound HRP.(d)Initial reaction rates of GO-bound HRP versus HRP concentration.Table 1sampleK m (mM)K cat (s -1)K cat /K m (mM -1s -1)Free HRP2.27161.7(34.1071.2GO Immobilized HRP (lower loading)1.96(0.2133.6(1.2017.1(1.22GO immobilized HRP (higher loading)1.76(0.1036.6(2.9020.8(0.52(28)Henriksen,A.;Schuller,D.J.;Meno,K.;Smith,A.T.;Gajhede,M.Biochemistry 1998,37,8504–8060.(29)Cooper,V.A.;Nicell,J.A.Water Res.1996,30,954–964.。

纳米酶标准术语作者：高利增梁敏敏温涛魏辉张宇范克龙江冰曲晓刚顾宁庞代文许海燕阎锡蕴来源：《中国科技术语》2020年第06期摘要：纳米酶是一类本身蕴含酶学特性的纳米材料，能够催化酶的底物，产生如同天然酶类似的催化反应，并具有酶促反应动力学等特征，属于一类新型模拟酶。

自2007年首次报道以来，纳米酶已成为多学科交叉的研究热点，其应用研究涉及医学、环境、农业、国防安全等多个领域。

近年来，基于纳米酶的新技术不断涌现，已有纳米酶相关产品问世。

此刻，十分有必要对纳米酶的相关术语进行研讨并形成规范，以便专业人员深入理解和准确评价纳米材料的类酶活性，也有助于促进纳米酶产业化。

关键词：纳米酶;术语;标准化中图分类号：N04文献标识码：ADOI：10.3969/j.issn.1673-8578.2020.06.004Abstract： Nanozyme refers to a class of nanomaterials that possess enzyme-like properties，capable of catalyzing the substrates of enzymes following the similar enzymatic kinetics. Nanozyme is as a new class of enzyme mimics which may be used as enzyme alternatives to improve human health. Since firstly reported in 2007， nanozyme has become an emerging field as a multidisciplinary research hotspot with broad application potential in many important fields such as biomedicine，environment treatment， agriculture and national security. With more and more novel nanozyme-based technologies and products developing， it is essential to make the related terms uniformed and standardized for nanozyme， which would not only be beneficial for scientists in this field to deeply understand and precisely evaluate nanozymes'catalytic activities， but also promote the industrialization of nanozyme.Keywords： nanozyme; vocabulary; standardization引言隨着纳米科技的发展及纳米生物医学研究的不断深入，人们发现很多纳米材料自身具有与天然酶类似的催化活性，能够在生理条件下催化天然酶的底物及其介导的生化反应，表现出类似的反应动力学和催化机理，将这类材料命名为纳米酶[1-2]。