Applications of inorganic nanoparticles as therapeutic agents

CHEMICAL INDUSTRY AND ENGINEERING PROGRESS 2017年第36卷第8期·3032·化 工 进展埃洛石纳米管在生物医学应用中的研究进展马智，李英乾，丁彤，董俊杰，秦永宁（天津大学化工学院，天津市应用催化科学与工程重点实验室，天津 300072）摘要：埃洛石纳米管（HNTs ）是一种新型的天然硅铝酸盐类纳米材料，具有独特的纳米管状结构及比表面积大、反应活性高等优点，近年来在生物医学运输领域中突显出愈来愈重要的应用价值。

本文简述了HNTs 的结构和性质，分析了HNTs 在生物医学领域应用的可行性，重点阐述了其在酶固定化、生物显像、生物支架、药物及基因靶向运输治疗等生物医学方面的研究和应用现状，并指出了在上述应用领域中HNTs 相对于传统无机纳米材料具有固载效果好、易于化学修饰、生物相容性高、毒性低、靶向选择性高等诸多优势。

最后指出了目前HNTs 在生物医学领域应用中还存在医疗成果转换周期长、成本效益高和药物释放作用机理不明确等挑战，并对其在疾病诊断、靶向递送药物及追踪治疗效果等一系列现代医疗技术的前景进行了展望。

关键词：埃洛石纳米管；载体；酶固定化；药物/基因靶向传递；生物成像中图分类号：O613.72；R945；Q5 文献标志码：A 文章编号：1000–6613（2017）08–3032–08 DOI ：10.16085/j.issn.1000-6613.2016-2296Research progress of halloysite nanotubes in biomedical scienceapplicationMA Zhi ，LI Yingqian ，DING Tong ，DONG Junjie ，QIN Yongning（Tianjin Key Laboratory of Applied Catalysis Science & Technology ，School of Chemical Engineering ，TianjinUniversity ，Tianjin 300072，China ）Abstract ：Halloysite nanotube （HNTs ） is a new natural aluminosilicate nanometer material. It has many advantages ，such as unique nano-tubular structure ，high specific surface area ，and high reactivity. In recent years ，HNTs has had more and more important applications in the field of biomedical transport . In this paper ，the structure and properties of HNTs were briefly introduced. The feasibility of HNTs application in biomedical field was analyzed. The researches and applications of HNTs in enzyme immobilization ，biological imaging ，biological scaffold ，targeted transport of cytotoxic drugs and gene ，and other aspects of the application ，were illustrated emphatically. Compared with traditional inorganic nanomaterials ，HNTs has many advantages in the field of biomedical applications such as good immobilization effect ，easy chemical modification ，high biocompatibility ，low toxicity ，and high selectivity. Finally ，the challenges of the HNTs in the field of biomedicine were pointed out ，including long medical results conversion cycle ，high medical costs ，and unexplained drug release mechanism. The prospects of the HNTs in a series of modern medical technologies ，such as disease diagnosis ，targeted delivery of drugs and follow-up treatment ，were also discussed. Key words ：halloysite ；support ；enzyme immobilization ；drug/gene targeting delivery; biological imaging近年来，有关生物医学领域的研究越来越受到人们的关注，尤其是基因改性及药物靶向治疗方面日益受到人们的重视[1]。

靶向抗肿瘤纳米药物研究进展论文摘要：靶向抗肿瘤药物特有的性质解决了传统的抗肿瘤药物的缺陷，使得抗肿瘤药物的进展到了一个新的阶段关键词：靶向抗肿瘤纳米肿瘤是当今严重威胁人类健康的三大疾病之一，而目前在临床肿瘤治疗和诊断中广泛应用的药物还多数为非选择性药物，体内分布广泛，尤其在一些正常组织和器官中也常有较多分布，常规治疗剂量即可对正常组织器官产生显著的毒副作用，导致患者不能耐受，降低药物疗效。

靶向制剂是以药物能在靶区浓集为主要特点的一大类制剂的总称, 属于第四代给药系统( drug delivery systerm, DDS) 。

靶向制剂给药后最突出的特点是利用药物载体系统将治疗药物最大限度地运送到靶区,使治疗药物在靶区浓集，超出传统制剂的数倍乃至数百倍,治疗效果明显提高。

减少药物对非靶向部位的毒副作用，降低药物治疗剂量并减少给药次数，从而提高药物疗效，这种治疗方法即被称为肿瘤靶向治疗。

现今在肿瘤靶向治疗领域，靶向抗肿瘤纳米药物研究正日益受到人们的普遍关注和重视，现就其近年来的研究进展综述如下。

1 靶向纳米药物的定义美国国家卫生研究院（NIH）定义：在疾病治疗、诊断、监控以及生物系统控制等方面应用纳米技术研制的药物称为纳米药物，其表面经过生物或理化修饰后可具有靶向性，即成为靶向纳米药物。

2 靶向纳米药物的特点基于纳米药物所特有的性质，决定了其在药物和基因运输方面具有以下几个优点：①可缓释药物，提高血药浓度，延长药物作用时间；②可减少药物降解，提高药物稳定性；③可保护核苷酸，防止其被核酸酶降解；④可提高核苷酸转染效率；⑤可建立新的给药途径。

而靶向纳米药物除这些固有优点以外，还具有：①可达到靶向输送的目的；②可在保证药物作用的前提下，减少给药剂量，进一步减少或避免药物的毒副作用等优点。

生物靶向纳米药物和磁性靶向纳米药物是目前靶向纳米药物研究的两大热点，并且都已具备了良好的研究基础。

3 靶向纳米药物的分类3.1被动靶向制剂微粒给药系统具有被动靶向的性能, 微粒的大小在011～3μm。

13Journal of Materials ScienceFull Set - Includes `Journal of Materials Science Letters'ISSN 0022-2461Volume 47Number 7J Mater Sci (2012) 47:3150-3158DOI 10.1007/s10853-011-6149-5Local structure and photocatalytic property of sol–gel synthesized ZnO doped with transition metal oxidesRongliang He, Rosalie K. Hocking &Takuya TsuzukiYour article is protected by copyright andall rights are held exclusively by Springer Science+Business Media, LLC. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If youwish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.13Local structure and photocatalytic property of sol–gel synthesized ZnO doped with transition metal oxidesRongliang He •Rosalie K.Hocking •Takuya TsuzukiReceived:4September 2011/Accepted:21November 2011/Published online:1December 2011ÓSpringer Science+Business Media,LLC 2011Abstract ZnO nanoparticles doped with up to 5at%of Co and Mn were prepared using a co-precipitation method.The location of dopant ions and the effect of doping on the photocatalytic activity were investigated.The crystal structure of nanoparticles and local atomic arrangements around dopant ions were analyzed by X-ray absorption spectroscopy.The results showed that the Co ions substi-tuted the Zn ions in the ZnO wurtzite phase structure and induced lattice shrinkage,while Mn ions were not com-pletely incorporated in the crystal lattice.The photocata-lytic activity under simulated sunlight was characterized by the decomposition of Rhodamine B dye molecules.It was revealed that Co-doping strongly reduced the photocata-lytic activity but Mn-doping showed a weaker effect on the reduction of the photoactivity.IntroductionZinc oxide is an important semiconducting material having a broad range of applications including transparent con-ductive oxides [1],ultraviolet (UV)light absorbers [2],and photocatalysts [3].Since the band gap energy of bulk ZnO crystals is around 3.3eV [4],UV rays with wavelength under 375nm can be absorbed by ZnO and hence ZnO hasbeen regarded as an excellent UV shielding material with a broad UV absorption characteristics and photo-fastness compared with other organic and inorganic UV shielding materials.The absorbed UV rays excite valence electrons onto the conduction band.When these photo-excited electrons and holes move to the particle surfaces where water and oxygen molecules reside,highly active free radicals such as super-oxide (O 2-)and hydroxyl (•OH)are generated and undergo secondary reactions such as the decomposition of organic compounds [5].This phenomenon called photocatalysis is useful for many applications,such as water-splitting,organic pollutant scavenging,and anti-fouling [6]and much effort was made to enhance the photocatalytic property of semiconductor nanoparticles [7–10].However,the degradation of organic molecules by the photocatalysis,including color fading [11],textiles aging [12],and even DNA and RNA damage [13],has been raising industrial and academic concerns for the safe use of ZnO as effective UV shielding agents.In order to decrease the photocatalytic activity,the gen-eration of free radicals needs to be suppressed.In general,two approaches can be considered for the reduction of pho-tocatalysis in semiconductor nanoparticles.The first approach is the surface coating on nanoparticles,which block the contact between excited electrons/holes and oxy-gen/water molecules [14].This approach has drawbacks as more materials are required to form barrier layers around particles which often results in higher production costs of the UV screening agents.The second approach is impurity doping to create chemical and,in some cases,physical defects in the crystal lattice that would act as the trapping and recombination sites of the excitons [15].In this study,transition metal atoms (Mn and Co)were introduced as impurities in ZnO crystallites to tailor the photocatalytic property.The ionic radii of Mn (0.066nm)R.He ÁT.Tsuzuki (&)Centre for Material and Fibre Innovation,Institute for Technology Research and Innovation,Deakin University,Geelong Technology Precinct,Geelong,VIC 3217,Australia e-mail:takuya.tsuzuki@.auR.K.HockingMonash Centre for Synchrotron Science,Australian Centre for Electromaterials Science and School of Chemistry,Monash University,Melbourne,VIC 3800,Australia123J Mater Sci (2012)47:3150–3158DOI 10.1007/s10853-011-6149-5and Co(0.058nm)are similar to that of Zn(0.060nm)and hence crystal doping of Mn and Co into the Zn sites is expected[16].They are also known to form deep energy levels within the bandgap,which may act as an efficient charge recombination sites[17,18].Different amounts of dopants,namely,1,2,3,and5at%were introduced to sol–gel synthesized ZnO crystalline nanoparticles.The doping process was extensively studied from raw materials tofinal products.X-ray absorption near edge structure(XANES) analysis was used to identify the oxidation states of dopant atoms.The local atomic arrangements and coordination states of doping elements were characterized by extended X-ray absorptionfine structure(EXAFS)analysis.The change in photocatalytic activity was analyzed via the decomposition of Rhodamine B(RhB)dye molecules under simulated sunlight in the presence of nanopowders. ExperimentalSample preparationCo-doped and Mn-doped ZnO was synthesized separately by using a sol–gel co-precipitation method to form doped zinc carbonate hydroxide and subsequent heat treatment to decompose zinc carbonate hydroxide into ZnO:Zn2þþOHÀþCO32Àþdopants!ðdoped)Zn4CO3ðOH)6ÁH2O,ð1ÞðDoped)Zn4CO3ðOH)6ÁH2O!ðdoped)ZnOþCO2þH2O:ð2ÞThe raw materials were Zn(CH3COO)2Á2H2O (Aldrich[99.0%),NaOH pellets(Chem-Supply97%), Na2CO3(Aldrich[99.0%),CoCl2Á6H2O(Aldrich[ 98.0%),and Mn(CH3COO)2(Fluka,98%),all of which were used without further purification.First,a mixed transparent solution-A of Zn2?and dopant ions with afixed atomic ratio(Dopant:Zn2?=1:99,1:49,1:32.3and1:19) was prepared in distilled water.At the same time,NaOH and Na2CO3were also dissolved in distilled water to prepare another solution-B which would be used as a precipitant.Then,the solution-A was added into the solution-B drop-wise with continued magnetic stirring at the speed of500rpm.The precipitate was immediately formed during the mixing of the two solutions.After1.5h of aging,the slurries were washed with distilled water several times using a centrifuge at the speed of7,000rpm (Eppendorf centrifuge5417R)until the salinity of the supernatant becomes less than100ppm.The washed powders were oven dried under80°C.Finally,the dried samples were heat-treated at400°C for1h to decompose doped Zn carbonate hydroxide into doped ZnO.Powder characterizationThe morphology of the synthesized particles was charac-terized by transmission electron microscopy(TEM)using a Philips CM-120microscope with the beam energy of 120kV.TEM specimen was prepared by evaporating a drop of the nanoparticle dispersion on a carbon-coated specimen grid.The crystal phase of the as-prepared particles was characterized by X-ray powder diffraction(XRD)mea-surement using an X-ray diffractometer(Panalytical X’Pert PRO MRD)with Cu K a radiation at a step width of0.02°. The operation voltage and current were set at40kV and 30mA,respectively.X-ray absorption spectroscopy(XAS)experiments were performed at the BL20B beamline(Australian National Beamline Facility)which was located in Photon Factory, Tsukuba,Japan.The samples were diluted with boron nitride to minimize the effects of self absorption and then filled in the5910mm2cells on the sample holder.The K-edge spectra of Zn,Mn,and Co elements were measured separately in afluorescence mode.The Average program [19]was used to average raw data.The EXAFS data analysis was carried out using the Ifeffit software package [20].Simultaneous thermogravimetric/differential scanning calorimetry(TG/DSC)measurements of undoped and doped zinc carbonate hydroxide were performed on a Netzsch STA409thermal analyzer to characterize the phase transformation during the heat treatment,using heating rates of10°C/min and nitrogenflowing rates of 100mL/min.The pyrolysed samples were also diluted with distilled water to0.01g/L at pH=7,which were used to measure the zeta potential using a Malvern Zetasizer Nano instrument.A Varian Cary3E spectrophotometer was used to measure the absorption spectra of the washed ZnO and doped ZnO powders at the wavelength range of 200–800nm.The actual doping levels in the powders were characterized using a Varian SPECTRAA-240Atomic Absorption Spectroscopy instrument in theflame mode. Photocatalysis testThe photocatalytic activity of the washed powders was measured by monitoring the degradation of RhB dye solution in the presence of the powder samples under simulated sunlight irradiated using an ATLAS Suntest equipment.For each measurement,0.012g of the powder sample was added into100mL of RhB aqueous solution having the concentration of0.0096g/L.The temperature of the solution was regulated to37°C and the simulated sunlight was irradiated up to3h.Three milliliters of the suspension was extracted every30min and then123centrifuged to separate the nanoparticles from the super-natant.UV–Vis absorbance spectra of the supernatant were measured with a Varian Cary3E spectrophotometer.The intensity of the optical adsorption peak around554nm, which is a characteristic absorption band of RhB,was used to monitor the rate of dye degradation.In order to assess the influence of powders’surface area on photocatalytic property,the specific surface area of the particles was analyzed by the Brunauer–Emmett–Teller (BET)gas absorption method using a Micromeritics Tristar 3000system.The result was also used to normalize the degradation rate of RhB.Results and discussionStructural and optical properties of doped ZnOThe synthesized ZnO nanopowders showed different colors at different doping levels.For Co-doped samples,the color changed from pure white,light green to dark green as the Co composition increased from0to5at%,while the color changed from pure white,yellowish to dark brown as the Mn composition increased from0to5at%.Figure1ashows a typical brightfield TEM image of undoped ZnO. It is evident that the powder consisted of agglomerated nanoparticles with the sizes between10to50nm.The TEM images of5at%Co-doped ZnO and5at%Mn-doped ZnO(Fig.1b,and c)showed similar particle sizes.It was observed that the undoped ZnO and other Co-doped and Mn-doped samples with different doping levels appeared nearly identical under TEM.The XRD patterns of synthesized undoped ZnO,Mn oxides,Co oxides,Co-doped ZnO and Mn-doped ZnO were shown in Fig.2.The XRD patterns of Mn oxides and Co oxides correspond to the JCPDS-ICDD index card No. 24-0734(Mn3O4)and42-1467(Co3O4),respectively.For undoped and doped ZnO,only the wurtzite phase corresponding to the standard crystallographic data in the JCPDS-ICDD index card No.36-1451was observed.No separated phase of dopants appeared was evident in the XRD pattern.Figure3shows the correlation between the nominal and measured doping levels for Co-and Mn-dopings.The real doping levels were closer to the nominal values for Co than Mn.Moreover,the Co-doping levels had a better linear correlation between real and nominal values.Hereafter,we use nominal dopant levels to identify the samples in the text.The UV–Vis transmittance spectra of undoped and doped ZnO are shown in Fig.4.As shown in Fig.4a and c, the increased doping level for both Co and Mn resulted ina Fig.1Bright-field TEM images of undoped and doped ZnO nanoparticles:a pure ZnO,b5at%Co-doped,c5at%Mn-dopedFig.2XRD patterns of undoped,Co-doped,and Mn-doped ZnO 123decrease in optical transmittance in the visible light range,while retaining good absorption property in the UV-light range.The bandgap energy of the samples were calculated by extrapolating the linear portion of the plot (a h m )2versus h m to the (a h m )2=0axis (Fig.4b,d)[21,22].The esti-mated bandgap energy of Co-doped and Mn-doped ZnO at low doping levels (1–3at%)were close or slightly larger than the value of pure ZnO (3.24eV),while the bandgap energy of 5at%Co-doped ZnO was lower than 3.24eV.This red-shift in bandgap energy,which was also observedby other groups [23,24],is mainly caused by the sp –d exchange interactions between the band-electrons and the localized d-electrons of the Co 2?ions at Zn sites [25].Location of dopantsXANES was carried out to investigate the location of the dopants ions within the Zn lattice.The possible locations of the dopant ions are:(1)the surface of the ZnO nanoparti-cles,(2)the Zn sites within the ZnO crystal lattice or (3)interstitial sites in the ZnO crystal lattice.Figure 5a shows the normalized Co K-edge XANES spectra of Co-doped ZnO,CoO,Co 3O 4,and a tetrahedral (T d )coordinated Co reference sample [Co(MeIm)2]n after the background was subtracted.The peak at ‘‘A’’in Fig.5a is usually referred to as a pre-edge peak and is assigned to a 1s ?3d transi-tion [26,27].The intensity of the peak A is reflective of the coordination environment;a tetrahedral cobalt compound will have higher intensity than an octahedral compound [28].This effect can be seen in Fig.5a,where the tetra-hedral (T d )reference had a substantially higher pre-edge peak than CoO in which Co is present in octahedral (O h )sites.This correlation indicates that the majority of doped Co-ions in ZnO were present at the tetrahedral sites,indicative of Co substituting Zn in the ZnO crystal lattice.XANES rising edge energy is indicative of oxidation states,which is also shown in Fig.5a as the peak ‘‘B’’.The rising edge energy positions of Co-doped ZnOsamplesFig.3The correlation between nominal and real dopinglevelsFig.4UV–Vis transmittance spectra and estimation of band gap energies of undoped,Co-doped,and Mn-doped ZnO:a UV–Vis transmittance spectra of undoped and Co-doped ZnO,b estimation of band gap energies of undoped and Co-doped ZnO,c UV–Vis transmittance spectra ofundoped and Mn-doped ZnO,d estimation of band gap energies of undoped and Mn-doped ZnO123were similar to each other and to that of the T d reference and CoO reference,indicating that the oxidation state of most of the Co ions in Co-doped ZnO systems were ?2.Figure 5b shows the Mn K-edge XANES spectra of Mn-doped ZnO,MnO,and Mn 3O 4.A pre-edge peak A is observed in all spectra,which is characteristic of transition metal ions [26,27].In the series of Mn-doped ZnO sam-ples,the positions of peaks/shoulders B and C did not shift when Mn-doping level increased.The peaks B and C of Mn-doped ZnO samples resemble in both energy and intensity to those of Mn 3O 4that contains both Mn 2?and Mn 3?as well as Mn T d and O h sites [29,30].This result indicates that not all the Mn ions substituted Zn sites in ZnO crystal lattice.In addition to the XANES data,the interpretation of EXAFS can also be used to elucidate the location of the Co ions.Figure 6shows k 3-weighted Co K-edge EXAFS andMn K-edge EXAFS compared with the Zn K-edge EXAFS for undoped ZnO.In Fig.6a,the shapes of EXAFS oscillation for Co-doped ZnO are nearly the same as that of undoped wurtzite phase ZnO but are quite different from those of CoO and Co 3O 4.This result suggests that the local atomic arrangement around Co ions in doped ZnO was similar to that of Zn in undoped ZnO.The increased noise in the Co 0.01Zn 0.99O spectrum at high K range is likely caused by self absorption phenomena [31].On the other hand,in Fig.6b,it is shown that the shape of Mn K-edge EXAFS k 3v (k )oscillation for all the Mn-doped ZnO samples were significantly different from those of Zn K-edge ZnO,Mn K-edge MnO,and Mn 3O 4.Ifonly the low K area (2–8A˚-1)is considered,the EXAFS oscillations of Mn-doped ZnO showed similar trend with that of Mn 3O 4,which suggests that the localatomicFig.5K-edge XANES spectra for Co-doped,Mn-doped ZnO,and reference samples:a Co-doped ZnO and reference samples at Co K-edge,b Mn-doped ZnO and reference manganese oxides samples at MnK-edgeFig.6k 3-weighted EXAFS oscillation functions;a Co K-edge for Co-doped ZnO,CoO,Co 3O 4,and Zn K-edge for undoped ZnO,b Mn K-edge for Mn-doped ZnO,MnO,Mn 3O 4,and Zn K-edge for undoped ZnO123arrangement around Mn ions in doped ZnO was similar to that of Mn in Mn 3O 4,which also correlates with the interpretation of XANES data,where the only difference between Mn 3O 4and Mn-doped Zn could be attributed to self absorption phenomena.The phase-corrected EXAFS k 3v (k )spectra were then Fourier transformed into radial distribution functions in R space to show the coordination states.For example,in the Fourier transforms of Zn K-edge EXAFS of undoped ZnO,the first peak at 1.9A˚corresponds to the first coordination shell with four oxygen atoms.The second peak at 3.3A˚represents the second coordination sphere,consisting of 12Zn atoms at the distance of 3.3A˚.In Fig.7a,the radial distribution functions of Co-doped ZnO samples,CoO and Co 3O 4around Co ions are compared with that of undopedZnO around Zn ions.The radial distribution functions of Co-doped ZnO samples around Co ions showed nearly identical peak positions with those of undoped ZnO around Zn ions,indicating that the majority of Co 2?ions substi-tuted the Zn 2?position in the ZnO crystal lattice [28,32],which supports the results of the XANES analysis descri-bed above.In Fig.7b,the radial distribution functions around Mn ions in Mn-doped ZnO,MnO,and Mn 3O 4samples are compared with that of undoped ZnO around Zn ions.For Mn-doped ZnO,the radial distribution functions around Mn ions were quite different from those around Zn ions in undoped ZnO and Mn ions in MnO.When dopant levels increased to 5at%,the second and third peaks gradually appearedat *2.63and *3.41A˚,respectively,which were similar to the peak positions of Mn 3O 4.Therefore,the majority of Mn ions did not replace Zn ions in the wurtzite crystal structure [24].This conclusion is consistent with a previous study by Erwin et al.[33]who found that the Mn 2?ion was difficult to substitute Zn 2?ions in wurtzite structure.The likely reason for the difficulty in Mn-doping is also attribute to the radii of the ions.The ionic radius of Mn is 0.066nm,larger than Zn (0.060nm)and Co (0.058nm).Hence,the substitution of Zn sites with Co ions is expected to occur more commonly than with Mn ions.Effects of doping processes on the location of dopants The process to form Co and Mn-doped ZnO consisted of two steps,namely (i)sol–gel co-precipitation of doped zinc carbonate hydroxide and (ii)thermal decomposition of doped zinc carbonate hydroxide into doped ZnO.In order to elucidate the effect of the synthesis routes on the loca-tion of dopant ions,the local structure of dopants ions in the zinc carbonate hydroxide was also studied by EXAFS.In Fig.8,the radial distribution functions of Mn-doped,Co-doped,and undoped zinc carbonate hydroxide at Mn,Co,and Zn K-edges,respectively,are shown.The radial distribution function of Co-doped zinc carbonate hydroxide has the shape and peak positions similar to that of undoped zinc carbonate hydroxide,indicating that the majority of Co 2?ions substituted the Zn 2?position in the crystal lat-tice of zinc carbonate hydroxide at the co-precipitation stage.On the other hand,the radial distribution function of Mn-doped zinc carbonate hydroxide has the shape and peak positions different from that of undoped zinc car-bonate hydroxide,suggesting that Mn 2?ions did not replace Zn 2?at the co-precipitation stage.Hence,the state of crystal doping before thermal decomposition appears to influence the successful replacement of Zn 2?ions with dopants in ZnO.Further pyrolysis analysis of as-prepared manganese carbonate hydroxide,cobalt carbonate hydroxide,zincFig.7Fourier transforms of the EXAFS k 3v (k )spectra:a Co K-edge radial distribution functions for Co-doped ZnO,CoO,Co 3O 4,and the Zn K-edge radial distribution function for undoped ZnO,b Mn K-edge radial distribution functions for Mn-doped ZnO samples,MnO,Mn 3O 4,and the Zn K-edge radial distribution function for undoped ZnO123carbonate hydroxide,Co-doped zinc carbonate hydroxide,and Mn-doped zinc carbonate hydroxide was shown in Fig.9which reveals the decomposition temperature of those compounds.For the as-prepared manganese carbon-ate hydroxide,cobalt carbonate hydroxide,and zinc car-bonate hydroxide,the decomposition temperature decreased gradually from 320,260to 225°C.For the as-prepared Co-doped zinc carbonate hydroxide,the ther-mal decomposition occurred between 225and 300°C and no separated decomposition stage was observed,indicating a uniform decomposition process.For the as-prepared Mn-doped zinc carbonate hydroxide,two decomposition stages were observed.The first stage at around 260°Crepresents the decomposition of zinc carbonate hydroxide with major mass loss.A slight mass loss appeared at around 350°C.The data suggest that inhomogeneous doping occurred during the precipitation process which has led to the formation of inhomogeneous phases after thermal decompo-sition,possibly consisting of undoped ZnO and Mn 3O 4.Furthermore,the results of zeta potential measurements in Fig.10provide another clue for the location of dopants.The zeta potential of as-synthesized Mn 3O 4and Co 3O 4by pyrolysis were -34.8and ?29.8eV,respectively.How-ever,the zeta potential of 5at%Co-doped ZnO was ?16.4eV which is very close to the value of undoped ZnO (?15.9eV)while the zeta potential of 5at%Mn-doped ZnO was only ?7.98eV.The lower zeta potential of Mn-doped ZnO may be caused by the negative zeta potential of Mn 3O 4that were precipitated on the particle surface,sug-gesting the possibility of Mn dopants concentrated on the particle surfaces (i.e.,surface doping).Photocatalytic activityFigure 11shows the doping effects on the BET specific surface area.The value of specific surface area does not differ much among the samples.For Co-doped ZnO,the surface area showed a general descending trend as the dopant level increased.For Mn-doped ZnO,it increased with dopant level.Figure 12a and b show the relative change in the intensity of the optical absorption peak of RhB as a func-tion of irradiation time.In the y axis,C is the absorbance value of RhB at each irradiation time interval at the wavelength of 554nm and C 0is the absorbance value before the irradiation when the adsorption and desorption equilibrium was achieved.The data was normalized using the BET specific surface area of each sample.It is evident that the photo-induced degradation of RhBwasFig.8Fourier transforms of the EXAFS spectra of zinc carbonate hydroxide,Co-doped zinc carbonate hydroxide,and Mn-doped zinc carbonatehydroxideFig.9TG curves of as-prepared manganese carbonate hydroxide,cobalt carbonate hydroxide,zinc carbonate hydroxide,Co-doped zinc carbonate hydroxide,and Mn-doped zinc carbonatehydroxideFig.10Zeta potential of undoped ZnO,Co-doped ZnO,Mn-doped ZnO,Co 3O 4,and Mn 3O 4123significantly slower in the presence of doped ZnO than undoped ZnO.Higher doping levels resulted in stronger reduction of RhB decomposition for both Co-doped ZnO and Mn-doped ZnO.Figure 12c also shows the calculated RhB degradation rate assuming the first-order kinetics [14,34].The doping level of 1at%induced a significant decrease in the photo-degradation rate,down to 24.3%of the original value for Co-doped ZnO and down to 66.6%for Mn-doped ZnO.When doping level was increased to 5at%,the photo-degradation rate decreased to 8.6%for Co-doped ZnO and to 33.3%for Mn-doped ZnO.The mechanism of decreased photocatalytic activity by successful lattice doping of Co in ZnO is considered to be not by creating physical defects but mainly by introducing deep band gap energy levels between the valence and conduction bands that would act as efficient recombination centers for photo-generated excitons [18,35].The recom-bination of electrons and holes inside the particles will give the charge carriers less chance to generate •OH and O 2-free radicals on particle surfaces [36].As for the case of Mn-doped ZnO,the cause of the reduction in photocata-lytic property may be attributed to the physical defects and increased oxidation state of Mn ions,which may act as trapping sites for photo-generated charges and promote the recombination of electron and holes and decrease the chance to generate •OH and O 2-free radicals on particle surfaces [37,38].ConclusionIn this study,a sol–gel co-precipitation method and sub-sequent heat treatment were used to synthesize ZnO nanopowders that are Co-doped and Mn-doped up to 5at%.Synchrotron X-ray absorption spectra and zeta potential measurements were used to investigate the locations of dopant ions in the nanopowders.It was shown that CoionsFig.11BET specific surface areas of undoped,Co-doped and Mn-dopedZnOFig.12Relative change in the intensity of the opticalabsorption peak at 554nm of RhB as a function of irradiation time:a with Co-doped ZnO,b with Mn-doped ZnO,c RhB degradation rate calculated assuming first-order kinetics123successfully replaced the Zn ions in the ZnO crystal lattice which may have created deep bandgap energy levels for the recombination of photo-generated electrons and holes.It was demonstrated that5at%Co-doping can effectively decrease the photocatalytic activity of ZnO down to\10% of the original reaction rate.Mn ions failed to substitute Zn ions in the ZnO crystal lattice and the majority of Mn may have precipitated on the particle surfaces.Nevertheless,the photocatalytic activity was also reduced by Mn-surface-doping with less efficacy than Co-lattice-doping. 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Intracellular Degradation of Multilabeled Poly(Ethylene imine)−Mesoporous Silica−Silica Nanoparticles:Implications for Drug ReleaseLotta Bergman,†,‡Pasi Kankaanpa a,§Silja Tiitta,§Alain Duchanoy,†Ling Li,†Jyrki Heino,§and Mika Linde n*,‡†Laboratory for Physical Chemistry,Åbo Akademi University,Porthansgatan3-5,FI-20500Turku,Finland‡Inorganic Chemistry II,University of Ulm,Albert-Einstein-Allee11,D-89081Ulm,Germany§Department of Biochemistry and Food Chemistry,Vatselankatu2,Arcanum thirdfloor,University of Turku,FI-20014Turku, Finland*Supporting Informationlinked to different regions of the particles in order to study thehuman SAOS-2cells.A novel,quantitative method for nanoparticlemicroscopy is applied.Our results suggest that the core−shell−shelloutside cells,which is of high importance for further application of thisuorescence microscopy,surface functionalizationTargeted delivery of drugs is one of the most promising approaches for the delivery of drugs associated with severe side effects,especially of importance for chemotherapy-based cancer treatment.1−3Different nanoparticle-based drug delivery systems have been shown to accumulate in tumors either by passive or active targeting and to be taken up by target cells. While passive targeting is based on nanoparticle accumulation in tumors through the leaky nature of the tumor vasculature, active targeting is based on the attachment of cell-specific ligands onto the nanoparticle surface that are recognized by receptors overexpressed on the target cells.4,5Nanoparticle-based drug delivery has several attractive features in addition to cellular targeting,the most important being the possibility to achieve high drug-loading levels and controlled release profiles.A promising new nanoparticle platform attracting wide current interest is based on amorphous mesoporous silica nanoparticles (MSNs).6−8MSNs can be synthesized with controlled particle sizes and shapes,9and the pore dimensions can be tuned within a range of some nanometers to tens of nanometers,allowing both small molecular drugs and proteins or genes to be accommodated inside the mesopores.10−12Furthermore,sur-face functionalization is relatively straightforward13−15which allowsfine-tuning of the drug−support and particle−bioenvir-onment interactions.In most cases,active targeting of MSNs has been demonstrated under in vitro conditions but has also been shown to work in vivo.16−19However,in order for a nanoparticulate carrier system to be effective,premature leakage of the drug before reaching the target cells has to be kept at a minimum.This is especially important for MSNs,as the release rate from carriers where the drug release is diffusion-controlled is highest initially and also often connected with an initial burst release.20The extent of drug leakage is naturally also dependent on the physicochemical properties of the drug, such as drug solubility(often pH-dependent),and the degradation rate of the MSNs,the circulation time before reaching the target cells.Much recent focus has been put on developing means for drug release that can be triggered by intracellular processes or by external stimuli.There are two Received:October13,2012Revised:February21,2013Accepted:March19,2013Published:March19,2013main approaches for achieving triggerable drug release;covalent linking of the drug to the support through cleavable bonds,or functionalization of the outer surface of the MSNs using sheddable coatings or coatings which change conformation upon environmental changes,typically pH,redox level,or temperature.Several in vitro studies have demonstrated that such strategies do indeed decrease the level of premature drug release and also allow for subsequent intracellular release of the cargo.21−24Examples are bonds that can be cleaved at pH values lower than that in the plasma,as is the case in intracellular compartments and also within the interstitial space of solid tumors and within inflammatory tissues.Recently,we studied the therapeutic efficiency of MSNs surface function-alized by hyperbranched poly(ethylene imine),PEI,to which the cancer drug methotrexate(MTX)had been covalently linked.25MTX is an antimetobolite which inhibits the enzymatic activity of dihydrofolate reductase(DHFR),thus blocking the biosynthetic pathway of nucleotides and proteins.26Here,MTX served both as a targeting ligand and the drug,as MTX is structurally very similar to folic acid,an often employed targeting ligand,and both molecules are taken up by similar cellular routes.However,MTX has to reach the cytoplasm in order to be therapeutically active.Based on our findings we tentatively suggested that MTX predominantly remained covalently attached to the particles when outside the cells,and that MTX detachment occurred readily after particle endocytosis.Several possible explanations could account for this observation,including particle degradation through silica dissolution,27−29detachment of the PEI layer to which MTX is attached from the MSNs after particle endocytosis by the cells, enzymatic degradation of the peptide bond30linking MTX to the particles once the particles are inside endosomal vesicles,or a combination of these.In any case,the kinetics of detachment remains an open question.Furthermore,it was recently suggested that MSNs are exocytosed by cancer cells and that these are subsequently taken up by other cancer cells,31and one important question is if there would still be drug molecules present in MSNs that can be released in such“second generation”cells or if all the cargo was already released in the cell offirst entry.PEI-coated MSNs have also been suggested to preferentially lead to intracellular release of hydrophobic drugs or drug models physically adsorbed into the mesopores of the MSNs.32Clearly,the answer to such questions is bound to be strongly system dependent,but a question is also how this could be studied experimentally.Here we present data where nonporous silica core−mesoporous silica shell particles have been synthesized with a PEI layer attached to the mesoporous shell of the particles,thus creating core−shell−shell MSNs.All three parts of the particle were covalently labeled with different fluorescent dyes so that the detachment of different parts of the particle could be studied and evaluated semiquantitatively.A three-layer model particle design was chosen to ensure that we would be able to track the main particle independently from that of the mesoporous portion of the MSNs,as nonporous silica is degrading at a slower rate than its mesoporous counterparts.Confocalfluorescence microscopy studies were carried out on human osteosarcoma SAOS-2cells as a function of incubation time,and the images were analyzed using the BioimageXD software,33allowing particles being located outside of the cells to be distinguished from particles internalized by the cells.The data suggests that,even after four hours,particles inside cells still do partly contain the PEI-fluorophore layer representing the drug model,while a fair amount of the drug model has been detached from the particles mainly in the form of a PEI-mesoporous silica complex.The study represents afirst step toward reaching a better understanding about the intracellular decomposition of functionalized MSNs.2.EXPERIMENTAL SECTION2.1.Synthesis of Three-Layer Fluorescent Silica Nanoparticles.The solid silica particle core was prepared based on the procedure described by Sto b er et al.34In a typical synthesis,250μg(1mg/mL)offluorescein isothiocyanate isomer I,FITC(minimum90%HPLC,Sigma-Aldrich),was mixed with3-aminopropyltriethoxysilane,APTS(Sigma-Aldrich),under inert atmosphere and added to an alkaline (ammonium hydroxide solution,max33%NH3,puriss.,Sigma-Aldrich)solution together with tetraehoxyothosilicate TEOS (purum≥98%GC,Fluka).The resulting synthesis mixture had molar ratios of0.1FITC:242APTS:4630TEOS:1892 NH4OH:129684H2O:266744EtOH.The solution was stirred overnight350rpm at RT.The mesoporous surface layer was then introduced based on the method described by Kim et al.35The nonporous silica nanoparticles were separated,washed carefully,and dispersed into basic reaction solution.The structure-directing agent CTAB(Sigma-Aldrich)andfinally TEOS together with tetramethylrhodamine isothiocyanate TRITC(Sigma-Aldrich)fluorophore conjugated with APTS were continuously added to the synthesis,here proceeding step by step according to the reference.The resulting solution had molar ratios of TRITC 0.01:APTS0.025:CTAB158:TEOS440:NH4OH2882:EtOH 152574:H2O615220.Synthesis was stirred overnight at500 rpm,and particles were separated,washed,and dried in vacuo at298K.To remove surfactant,particles were extracted under sonication;30min in acidic ethanol(1:8-mixture of HCl and absolute ethanol).This treatment was repeated three times to ensure complete surfactant removal.Particles were further separated by centrifugation and carefully vacuum-dried over-night at298K.The so-synthesized core−shell particles were further surface-modified by hyperbranching polymerization of polyethyleneimine(PEI),using aziridine as a precursor. Aziridine was synthesized from aminoethylsulfuric acid (Sigma-Aldrich,Miss,USA)according to the procedure described by Allen et al.36The surface polymerization of PEI was performed in one step under argon as the protective gas. Particles were dispersed in toluene,and catalytic amounts of acetic acid were added,after which aziridine was added.In a typical conjugation,35μL of aziridine for100mg particles was used.The suspension was refluxed under stirring overnight at 348K,filtered,washed,and vacuum-dried at298K.The third fluorophor,Alexa633,was linked to the particle described above by attaching it to the surface amino groups.In a typical procedure,100mg of particles were dispersed in toluene. Surface primary amino groups were activated with DIPEA(100μL,1μL/mL in DMF)and further conjugated with Alexa633fluorophore(200μL,1μL/mL in DMF)under shaking for2h at RT(298K).Finally the particles were separated,washed with ethanol,and vacuum-dried at298K.2.2.Particle Characterization.The structure of the nanoparticles was confirmed by low angle,main reflection at 2.2°2Θ,powder-XRD using a Kratky compact small-angle system(Hecus Braun,Austria),Seifert ID-300X-ray generator with maximum intensity of50kV and40mA,and sample-to-detector distance of267mm.Thermogravimetric analysis was performed in air with a Netzsch STA449C cell setup with a heating rate of10K/min. Dynamic light-scattering(DLS)and zeta-potential measure-ments were performed using a Nano ZS(Malvern,Worcester-shire,UK)setup in a HEPES buffer.Measurements were performed at298K,using a monochromatic laser,with a working wavelength of632.8nm and using non-invasive back-scatter(NIBS),with the detector positioned at173°relative to the laser beam.Scanning electron microscopy(Jeol JSM-6335F,Jeol Ltd., Japan)was performed using an acceleration voltage of10kV and working distance of9.6mm.Transmission electron microscopy(TEM)micrographs were measured using a Zeiss Libra120TEM setup operated at80 kV.Nitrogen adsorption−desorption experiments(ASAP2010 sorptometer,Micromeritics)were carried out at77K.All samples were degassed for8h at323K before measurements. The specific surface area was determined by the BET method, and the pore dimensions were determined using the BJH method(desorption branch).2.3.Confocal Imaging and Image Analysis/Quantifi-cation.Particles were dispersed(1mg/mL)in water one hour before their delivery to cells.As silica particles do not hold intact in aqueous solvents and a relatively fast process of dissolution is reported for contact times below1h,the delay has to be taken into account when interpreting the imaging results.In this study,we concentrate on what happens to the particles in in vitro conditions at the exposion time point and1 and4h thereafter.Human SAOS-2cells were incubated in Dulbecco’s modified Eagle’s medium with the particles at a concentration of125μg/ mL.The cells werefixed with4%paraformaldehyde(20min RT)after5min,1h,or4h,and embedded in Mowiol. Thickness-selected cover glasses(0.17±0.01mm/Assistent) were used to minimizefluorescence intensityfluctuations.2D confocal images were acquired with a Zeiss AxioObserver Z1 (objective63x,1.4)equipped with LSM510,both near the glass surface and from the upper half of the cell.All three wavelengths(488,543,and633nm)were excited and recorded separately(multitracking)line-by-line,with pinhole-calibrated dichroics andfilters optimized for minimum bleed-through,and pinholes were adjusted for an optical slice thickness of700nm and pixel size of94nm×94nm for every wavelength. Acquisition settings and conditions were kept as constant as possible to enable the comparison offluorescence intensities. Importantly,images were always acquired with the same pixel density,roughly following the Nyquist theorem,and the detector sensitivity and background offset were kept constant at such values that the whole intensity range of the samples was recorded without saturation.Regions of interest(ROI)were drawn onto the images so that particles outside cells were analyzed separately from particles inside cells.In total approximately40images from inside and40from outside the cells were analyzed for each time point.Segmentation-based analyses were done byfirstfiltering the images with hybrid median2D and Gaussian smoothing (dimensionality3:4,4,2),then thresholding them for maximum object number(minimum object size10)andfinally running object separation(level1,image spacing used).The segmented objects were then analyzed for the intensities of each of the three wavelengths as well as average object size. Colocalization studies were performed to confirm the relation-ships of the different colors inside the cells,and all pixels in the images were analyzed,unlike in segmentation-based analyses. Colocalization analyses were done by using automatic thresh-olding and then recording all six possible Manders’colocalization coefficients,and observing changes in these between the three time points;5min,1h,and4h.All image analysis was done with the BioImageXD software.33The results were statistically analyzed with a t test for unequal variances and sample sizes,and significance was marked onto graphs as follows:ns=p>0.05,*=p<0.05,**=p<0.01,***=p<0.001.■RESULTS AND DISCUSSIONAn scanning electron microscopy(SEM)image of the core−shell−shell MSNs is shown in Figure1a.The particles have a diameter of about240nm and have a very narrow size distribution.A TEM image of a native silica core−shell MSN is shown in Figure1b.The core−shell structure of the MSNs is clearly seen,and the mesoporous shell is uniform.From the TEM image a mesoporous silica layer thickness of about12nm can be estimated,which corresponds to about30%of the total volume of the MSN.A nitrogen sorption isotherm measured for the native core−shell MSNs is shown in Figure2.A pronounced uptake at a relative pressure close to0.3p/p o is characteristic for MSNs synthesized using C16TAB as a structure-directing agent and corresponds tofilling of mesopores with a diameter of about4nm with a narrow pore size distribution.The BET specific surface area was344 m2/g,and the specific pore volume of the primary mesopores was0.34cm3/g,which is slightly higher than that expected from the relative contributions of the mesoporous surface layer and the solid core,suggesting that the thickness of the surface layer based on TEM might be underestimated.The pronounced uptake at higher p/p o is due tofilling of interparticulate porosity.The low-angle XRD pattern(Figure 3c)exhibited a main reflection at2.2°2Θand additional intensity in the regions expected for(11)and(20)reflections of a2D hexagonal mesophase,suggesting that the mesoporous surface layer is not disordered but has a structural motif similar to that observed for corresponding MSNs void of the solid core.Assuming a2D hexagonal arrangement of pores,the lattice spacing derived from the(10)reflection is4.83nm. Before PEI functionalization,the core−shell MSNs have a zeta-potential of−27mV in25mM HEPES pH7.2,which increases to+52mV after PEI functionalization,reflecting successful attachment of the highly cationic PEI layer.Thermogravimetric analysis shows a mass loss of7.1wt%in comparison to reference particles lacking PEI.Upon attachment of the Alexa 633dye to PEI,the zeta-potential increased slightly to55mV. The particles could be fully dispersed in HEPES,as evidenced by dynamic light-scattering(Figure S1).Thefluorescence emission colors of the different parts of the core−shell MSNs are schematically shown in Figure1a.The total amounts of fluorophores covalently attached to the PEI-MSNs were analyzed by dissolving the particles in2M NaOH-solution1 mg/mL and analyzing the dye content in the supernatant by fluorescence spectrometry.The PEI-MSNs contained Alexa633 1.09μg/mg particles(attached to the PEI layer),TRITC0.12μg/mg particles(attached to the mesoporous silica layer),and FITC0.04μg/mg particles(attached to the nonporous silica core).Fluorophore leakage was studied under in sink conditions (particle concentration75μg/mL)in HEPES buffer(pH7.2) at37°C,and no leakage of FITC or Alexa633was observed,but about 10%of the TRITC fluorophore was detected in the supernatant after the 4h incubation time.It was also ensured that the free fluorophore,de fined by the maximum concentration of FITC used in the study,could not be detected by confocal fluorescence microscopy under the applied imaging conditions.In the presence of cells no clumps or clusters of free fluorophore could be detected,and thereforeany detectable fluorescence signals could positively be attributed to fluorescence originating from the particles.While Alexa633and TRITC show pH-independent fluorescence intensities in the pH range of interest in our study (pH about 5−7.2),the fluorescence of FITC is known to be strongly pH dependent due to the p K a of FITC of 6.4.Tests using free FITC in bu ffers showed a decrease in the fluorescence intensity of about 50%at pH 6.5and of about 85%at pH 5.5as compared to the intensities measured for the same FITC concentration at pH 7.5,in good agreement with literature values.37Performing similar experiments using the core −shell particles void of PEI resulted in a FITC fluorescence intensity decrease of 60%at pH 5.5as compared to pH 7.5;that is,the decrease in emission intensity was lower than that of the free fluorophore.The smaller pH dependency of the FITC fluorescence emission intensity when incorporated into Sto b er-type silica is tentatively attributed to di fferent local pH values experienced by the probe inside the Sto b er particles(silicaFigure 1.Three color core −shell particles.(a)Schematic representation of the particle structure.Fluorescent dyes are covalently linked inside the silica network and likewise covalently linked to the surface polyethylene imine amino groups.(b)Scanning electron microscope image (SEM),scale bar corresponds to 1μm.(c)Transmission electron microscope image (TEM),scale bar corre-sponds to 20nm.Figure 2.(a)SAXS graph shows the ordered porosity of the mesoporous surface layer.(b)Nitrogen physisorption isotherm for prepared core −shell particles.(c)Nitrogen physisorption BJH analysis shows narrow pore size distribution in the mesoporous layer.contains Bro s tedt acidic silanol groups),38but the results also highlight that the Sto b er particles are not completely nonporous,as external pH changes can indeed be felt by the dye.Importantly,no pH-dependent changes in the fluorescence intensity of FITC were observed for PEI-coated core −shell particles,suggesting that the presence of PEI bu ffered the pH inside the core −shell particles.These results will be discussed below as a means for investigating the detachment kinetics of PEI from the MSNs,in addition to the analysis of the locus of the di fferent fluorescent dyes.For clarity,the di fferent fluorescence emission colors will be indicated together with their locus in the particles as follows;green (C)for FITC-core,yellow (MP)for TRITC-mesoporous shell,and red (PEI)for Alexa 633-PEI.3.3.Confocal Image Analysis. 3.3.1.Segmentations.Representative segmentations of the SAOS-2cells as a function of particle incubation times ranging from 5min to 4h are shown in Figure 3.The images were acquired in such a way that it could be distinguished whether fluorescence was originating from particles located outside or inside the cells.When close to glass surface (but still slightly above it)and inside the cell perimeter,one can specify from the 3D imaging characteristics of the confocal microscope the objects located inside and outside the cell.The same is true higher up,close to the summits of the cells,when images were taken slightly below the summit and also inside the cell perimeter.The cell surface is clearly identi fiable in the microscopy images shown in Figure 3,and regions of interest (ROI)were drawn to allow independent analyses of intracellular and extracellular regions.As can be seen in Figure 4(5′),MSNs are internalized already at the 5min time point,and the number of internalized MSNs increaseswith time.The MSNs are compartmentalized inside the cells,in agreement with the well-established endosomal uptake of this type of particles,while particles located outside the cells appear to be well-dispersed.2D image analysis of particle cluster size inside and outside of the cells (Figure 5)appreciates a decrease of cluster size inside the cells with time.Here,cluster sizes de fined by confocal microscopy are to be evaluated for relative di fferences only,not as absolute values,because of light scattering as de fined by the point spread function.Fast initial (5′)internalization leadstoFigure 3.Typical examples of the confocal fluorescence microscopy images analyzed,here from the green channel at the 1h time point.Left:original images,middle:original images with regions of interests (ROIs)drawn,right:segmentation results within the ROIs.Regions marked with A:segmented objects analyzed as intracellular.Regions marked with B:segmented objects analyzed as extracellular.Image intensities have been enhanced by linear intensity transfer functions to improve the visualization.Upper row:Image taken close to the upper part of the cells.Lower row:Image taken close to the glass substrate.The scale bar corresponds to 10μm.Pseudocolored versions of all of the three fluorescence channels observed is available as Supporting Information for the image in the lowerrow.Figure 4.Confocal images taken from the yellow channel from the top part of thecell.Figure 5.Segmented object size (pixels)from the yellow channel,inside cells (■)and outside cells (▲)as a function of incubation time.The analysis is based on the yellow channel (TRITC-MP),as it showed highest fluorescence intensity,but qualitatively similar results were obtained also when performing the analysis based on the green or the red channel.high particle concentration in intracellular compartments.Our earlier internalization studies with SAOS-2cells show an increase in integrin cluster size up to 45min,whereafter the cluster size starts to decline.39Similarly,in the present study,the point measurement at 1h shows a decrease of intracellular cluster size.Confocal images from the yellow channel at all three time points (Figure 4)support the numerical data:initially (5′)particles are seen to form larger clumps.At the 1h time point,the clusters are smaller and fainter,and they are perhaps closer to the nucleus as internalization has progressed.This is a trend that continues further at the 4h time point.A more detailed discussion about the potential locus of MSNs as a function of time is given below,in connection with a more detailed analysis of the time-dependent fluorescence measure-ments of the di fferent colors.All three fluorescent colors were segmented separately,and the absolute intensity of each fluorescent color was plotted over time.Interestingly,outside the cells no signi ficant changes in the fluorescence intensities of single fluorophores from individual channel-spesi fic segmentations were observed (Figure 6a).Additionally,the fluorescence intensities of all colors in relation to the yellow channel were calculated (Figure 6b).Intensities of all colors were then measured based on the yellow channel segmentations;that is,pixels having yellow fluorescence were used as the basis of the total intensity of the green and red fluorescence,and the intensities were normalizedagainst the total yellow fluorescence intensity.Also in these measurements no signi ficant changes in the dye intensities outside the cells over time were detected.This suggests that particles located extracellularly remained stable or dissolved in a homogeneous manner.The time-dependency of the fluores-cence intensity measured for the three di fferent fluorophores originating from particles located inside the cells followed a distinctively di fferent pattern,as shown in Figure 6c and d.While the total fluorescence emission intensity of the yellow (MP)remained virtually constant over time,a clear decrease in the corresponding total intensities of the red (PEI)and green (C)was observed signi ficantly with increasing incubation time.The decrease in the red (PEI)intensity,both in the individual segmentations and when normalized against the intensity of the yellow fluorescence,is consistent with a partial detachment of PEI from the particles with time together with dilution of the detached PEI,possibly due to endosomal escape.40,41This leads to a decrease of the red fluorescence observed in these pixels to values below the set detection limits.The decrease in the green (C)fluorescence intensities with time supports this suggestion,due to the observed decrease in the FITC fluorescence emission intensities with decreasing pH in the absence of pH-bu ffering PEI on the particles.3.3.2.Colocalization Analyses.Colocalization analyses of the three di fferent fluorophores were carried out in order to get a more detailed picture of the particle degradation process.TheFigure 6.Intensity of the segmented objects over time,measured both inside and outside the cells.(a)Separate segmentations outside the cells.(b)All colors segmented in relation to yellow channel outside the cells.(c)Separate segmentations inside the cells.(d)All colors segmented in relation to yellow channel inside the cells.[ns]indicates nonsigni ficant change,and stars (*),up to three,describe the degree of a signi ficant change.(yellow ■,red ●,green ▲).results are expressed as Manders’coefficients,always for two colors at a time.First,the number of pixels that contain both colors are determined,and the total intensity of a given color within these pixels is divided by the total intensity of one or the other color within that segment.Thus,the Manders’coefficients of,for example,red toward yellow and yellow toward red,are not necessarily the same.For example,red could be completely colocalized with yellow,which would correspond to a Manders’coefficient of1,while there could be yellow pixels which would not coincide with pixels showing both red and yellow,thus resulting in a Manders’coefficient smaller than1.It should also be noted that the determined Manders’coefficients are more sensitive to the stable particles as compared to potentially detachedfluorophores,as detached fluorophores are diluted and may fall below the set threshold values.This also implies that intracellular analyses are more sensitive to colors remaining inside cellular compartments rather than being located in the cytoplasm for the same reasons. The analyses of the Manders’coefficients were carried out separately for particles located inside or outside of the cells. Interestingly,the changes in the Manders’coefficients for particles located outside the cells was within experimental error for the time-period studied(data not shown),indicating that the particles contained allfluorophores up to the time-point of 4h.Importantly,this does not mean that there was no particle dissolution,but it indicates that the particles did at least not completely disintegrate nor did any of the layers completely detach from the main particle during the time of observation. Inside the cells,the situation was quite different.The Manders’coefficients of all possible color-combinations inside the cells are shown in Figure7a−c.Depending on the combinations,the Manders’coefficients,that is,the colocalization of different colors,are remarkably different when followed as a function of incubation time.The Manders’coefficient for green(C)−yellow(MP)remain high over the time of observation,while the corresponding values for yellow(MP)−green(C)decrease significantly over time.This suggests that the mesoporous silica layer is detaching from the main particles,leaving some of the mesoporous layer behind.Also the Manders’coefficient for red(PEI)/yellow (MP)remain high throughout the experiment,while the Manders’coefficient for yellow(MP)/red(PEI)decreases significantly over time.This result is consistent with a dissolution process where the mesoporous layer is dissolved in a way that some of the mesoporous silica layer is detaching together with the PEI layer from the particles,while a portion of the mesoporous silica layer is still associated with the main particle.This is also seen in the high Manders’coefficient for green(C)/yellow(MP)throughout the experiments.Interest-ingly,the corresponding Manders’coefficients for red(PEI)/ green(C)and green(C)/red(PEI)cannot be explained using this simple dissolution model.The Manders’coefficients indicate that the colocalization of green(C)/yellow(MP) remain high throughout the experiment,as expected based onthe values of the discussed Manders’coefficients,while significantly decreasing colocalization is seen in the decreasing values of the Manders’coefficient for yellow(MP)/green(C) with time.This suggests that the core is separating from the PEI layer,while the PEI layer remains on the Sto b er particle core.Thisfinding can be explained by a coexistence of particles where some fraction of the particles have lost the PEI layer, while some particles still have a PEI layer on them.As discussed above,the detachment of PEI,also supported by the time-dependent totalfluorescence intensity analysis,together with the escape of PEI into the cytoplasm,will lead to an overestimation of the amount of PEI present in intracellular compartments.Here PEI may be attached to particles or to fragments of mesoporous silica still being present within intracellular compartments.However,the results clearly suggest that the PEI layer is detaching from the particles,most probably both in the form of free PEI and PEI attached to fragments of mesoporous silica,and that this process preferentiallyoccurs Figure7.(a)Manders’colocalization coefficients as a function of incubation time(red/green■and green/red⧫),(b)Manders’colocalization coefficients as a function of incubation time(green/ yellow■,yellow/green⧫),and(c)Manders’colocalization coefficients as a function of incubation time(red/yellow■,yellow/ red⧫).。

基于SERS技术快速实现现场毒品检测孟娟;杨良保;张莉;唐祥虎【摘要】本文通过对尿液进行前处理,实现了人体尿液中毒品快速分离和纯化.并且,我们以自组装的金纳米棒为SERS基底对纯化的尿样进行检测,结合便携式拉曼光谱仪成功实现了对尿液中冰毒、摇头丸、甲卡西酮的分析检测,具有很高的灵敏性.整个纯化和检测过程只需要～3.5 min,该方法方便、快捷,有望能实现现场对吸毒人员尿样中毒品的灵敏性检测.【期刊名称】《光散射学报》【年(卷),期】2016(028)004【总页数】5页(P297-301)【关键词】表面增强拉曼光谱;便携式拉曼光谱仪;毒品检测;尿液【作者】孟娟;杨良保;张莉;唐祥虎【作者单位】安徽大学化学化工学院,合肥230039;中国科学院合肥智能机械研究所,合肥230031;中国科学院合肥智能机械研究所,合肥230031;安徽大学化学化工学院,合肥230039;中国科学院合肥智能机械研究所,合肥230031【正文语种】中文【中图分类】O657.37冰毒(methamphetamine,MAMP)、摇头丸(3,4-methylenedioxy methamphetamine,MDMA)、甲卡西酮(methcathinone,MCAT)是现如今的新兴合成类毒品,毒品的滥用、成瘾与流行,已成为当今世界日益严峻的问题。

根据《2014年中国毒品形势报告》,目前我国以冰毒、摇头丸、甲卡西酮等为主的合成毒品滥用人员增长迅速,吸毒的人员出现低龄化、多元化,且合成毒品滥用群体比例已经超过海洛因。

毒品打乱了社会和谐,破坏了家庭的稳定。

因此,对毒品的检测和抑制具有非常重要的意义。

常见的人体内毒品检材有血液、唾液、汗液、头发和尿液等。

但是由于生物检材成分比较复杂,存在大量的干扰物,直接对其分析检测有很大的难度。

目前,常见的毒品检测方法主要有气相色谱法(GC)[1]、高效液相色谱法(HPLC)[2]、薄层层析法、气相色谱—质谱联用法(GC-MS)[3]、液相色谱—质谱联用法(HPLC-MS)[4]、胶体金法等分析方法。

化学主题学术英语作文篇一The application of chemistry in environmental protection has become an increasingly critical area of research, particularly as global environmental challenges such as climate change, pollution, and resource depletion continue to escalate. The integration of chemical principles and technologies into environmental science has led to innovative solutions that not only mitigate environmental damage but also promote sustainable development. This paper aims to explore the multifaceted role of chemistry in addressing environmental issues, with a particular focus on green chemistry, catalytic processes in wastewater treatment, and the development of novel materials for carbon emission reduction.Green chemistry, often referred to as sustainable chemistry, is a revolutionary approach that seeks to design chemical products and processes that minimize the generation of hazardous substances. Unlike traditional chemical practices, which often prioritize yield and efficiency at the expense of environmental health, green chemistry emphasizes the use of renewable resources, energyefficient processes, and nontoxic reagents. For instance, the development of biobased plastics derived from renewable biomass, such as cornstarch or sugarcane, has significantly reduced reliance on petroleumbased plastics, which are notorious for their persistence in the environment and contribution to microplastic pollution. Furthermore, green chemistry has enabled the synthesis of pharmaceuticals and agrochemicals with reduced environmental footprints, as exemplified by the use of enzymatic catalysis, which operates under mild conditions and generates fewer byproducts compared to conventional chemical synthesis.Catalysis, a cornerstone of modern chemistry, plays a pivotal role in environmental remediation, particularly in the treatment of industrial wastewater.Industrial effluents often contain a complex mixture of organic pollutants, heavy metals, and toxic compounds that pose significant risks to aquatic ecosystems and human health. Advanced oxidation processes (AOPs), which utilize highly reactive species such as hydroxyl radicals to degrade pollutants, have emerged as a powerful tool for wastewater treatment. For example, the use of titanium dioxide (TiO2) as a photocatalyst in AOPs has demonstrated remarkable efficiency in breaking down persistent organic pollutants, such as dyes and pharmaceuticals, into harmless byproducts. Moreover, the integration of nanotechnology with catalysis has led to the development of highly efficient and selective catalysts that can operate at lower temperatures and pressures, thereby reducing energy consumption and operational costs. The application of catalytic converters in automotive exhaust systems is another notable example, where platinumgroup metals catalyze the conversion of harmful nitrogen oxides (NOx) and carbon monoxide (CO) into less toxic nitrogen (N2) and carbon dioxide (CO2).The development of novel materials with tailored properties has opened new avenues for reducing carbon emissions and enhancing energy efficiency. Carbon capture and storage (CCS) technologies, which aim to capture CO2 emissions from industrial sources and store them underground, rely heavily on advanced materials such as metalorganic frameworks (MOFs) and zeolites. These materials exhibit high surface areas and tunable pore sizes, enabling the selective adsorption of CO2 from flue gases. For instance, MOFs composed of zinc ions and organic linkers have shown exceptional CO2 adsorption capacities, making them promising candidates for largescale CCS applications. Additionally, the advent of perovskite solar cells, which utilize hybrid organicinorganic materials, has revolutionized the field of photovoltaics by offering higher efficiencies and lower production costs compared to traditional siliconbased solar cells. These materials not only contribute to reducing greenhouse gas emissions but also pave the way for a transition to renewable energy sources.In conclusion, the application of chemistry in environmental protection is a testament to the discipline's transformative potential in addressing some of the mostpressing challenges of our time. Through the principles of green chemistry, the development of advanced catalytic processes, and the innovation of novel materials, chemistry offers a diverse array of tools and strategies for mitigating environmental degradation and promoting sustainability. As the global community continues to grapple with the consequences of industrialization and urbanization, the role of chemistry in shaping a more sustainable future cannot be overstated. It is imperative that ongoing research and collaboration across disciplines are encouraged to further harness the power of chemistry in safeguarding our planet for future generations.中文翻译:化学在环境保护中的应用已成为一个日益重要的研究领域，尤其是在全球环境挑战如气候变化、污染和资源枯竭不断加剧的背景下。

细菌生物膜的防治进展丁进亚;黄前川【摘要】生物膜是细菌对抗不利环境、导致持续感染和耐药性的重要方式,常常给临床治疗带来极大困难.生物膜的形成受到多种因素的影响,包括生物医学材料、细菌的群体感应信号、细胞外多糖、二价阳离子浓度、环鸟苷二磷酸信号途径等.新型抗菌生物材料的研制和细菌生物膜形成机制的阐明,为防治细菌生物膜引起的难治性感染提供了新途径.%One major cause for persistent infection and drug resistance is the capability of bacteria to grow in biofilms that protects them from adverse environmental factors, resulting in great difficulties in clinical treatment. Biofilm formation regulated by many factors include biomedical materials , bacterial quorum sensing signals , extracellular polysaccharide , divalent cation concentration , and c-di-GMP signal pathway. Further researches on bacterial biofilm formation and new antimicrobial biomaterials maybe provide novel therapeutic pathwaya for refractory infections.【期刊名称】《医学综述》【年(卷),期】2011(017)003【总页数】3页(P452-454)【关键词】生物膜;抗菌生物材料;群体感应;细胞外多糖【作者】丁进亚;黄前川【作者单位】广州军区武汉总医院检验科,武汉,430070;广州军区武汉总医院检验科,武汉,430070【正文语种】中文【中图分类】R378.1生物膜是微生物细胞(如细菌、真菌、原虫)及其产生的细胞外大分子多聚物所形成的一种特殊细菌群落，具有高度的组织化。

综述结论部份的写法和范文-by zhenmafudan from emuchby zhenmafudan from emuch也曾读过很多综述，感到很多综述虎头蛇尾，引言部分牛皮吹得很震天响，可是到了最后越写越粗糙，到了结尾部分竟然几句话草草收尾了！读了如此综述，感到通篇就是“谁谁谁做了什么，他们发现了什么”，只有罗列事实，没有评述。

正如K.R. Seddon在Inorganic Liquids in Synthesis (Second Edition)一书的序言中所说："How many papers within this annual flood of reviews say anything critical, useful, or interesting? How many add value to a list of abstracts which can be generated in five minutes using SciFinder ot the ISI Web of Knowledge? How many of them can themselves be categorised as garbage? It is the twenty-first century----if a review is just an uncritical list of papers and data, what is its value?"经过长期总结，发现综述的结论部份其实是有某种“套路”的，正如中国古代的词，是有套路的一样！写综述的结尾部分如同以前高中回答政治题目，要“回答到点子上”。

综述的结论部份要实现几种基本功能、基本内容：第一层次的内容就是要总结上面已经综述了什么东西，简要地给读者进行“复习”，然后说这些学术进展有什么意义。

第二层次的功能是“打补丁”。

任何综述都不可能面面俱到，往往读者看了文章会说：“为什么我的文章没有被引用？”于是就要在综述里实现把“补丁”打好。

Hollow silica nanoparticles loaded with hydrophobic phthalocyanine for near-infrared photodynamic and photothermal combination therapyJuanjuan Peng a,Lingzhi Zhao a,Xingjun Zhu a,Yun Sun a,Wei Feng a,*,Yanhong Gao b, Liya Wang c,**,Fuyou Li a,*a Department of Chemistry&State Key Laboratory of Molecular Engineering of Polymers,Fudan University,220Handan Road,Shanghai200433,PR Chinab Department of Geriatrics,Xinhua Hospital of Shanghai Jiao Tong University,School of Medicine,Shanghai200092,PR Chinac College of Chemistry and Pharmaceutical Engineering,Nanyang Normal University,Nanyang473061,PR Chinaa r t i c l e i n f oArticle history:Received16May2013 Accepted8July2013Available online24July2013Keywords:PhthalocyanineHollow silica nanoparticles Photothermal therapy Photodynamic therapy a b s t r a c tOwing to the convenience and minimal invasiveness,phototherapy,including photodynamic therapy (PDT)and photothermal therapy(PTT),is emerging as a powerful technique for cancer treatment.To date,however,few examples of combination PDT and PTT have been reported.Phthalocyanine(Pc)is a class of traditional photosensitizer for PDT,but its bioapplication is limited by high hydrophobicity.In this present study,hollow silica nanospheres(HSNs)were employed to endow the hydrophobic phthalocyanine with water-dispersity,and the as-prepared hollow silica nanoparticles loaded with hy-drophobic phthalocyanine(Pc@HSNs)exhibits highly efficient dual PDT and PTT effects.In vitro and in vivo experimental results clearly indicated that the dual phototherapeutic effect of Pc@HSNs can kill cancer cells or eradicate tumor tissues.This multifunctional nanomedicine may be useful for PTT/PDT treatment of cancer.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionPhototherapy has attracted much interest in recent years as a powerful technique for cancer treatment due to the convenience and minimal invasiveness.Photodynamic therapy(PDT)and pho-tothermal therapy(PTT)are two typical phototherapy approaches, which require light absorption and photosensitizer to generate reactive oxygen species and heat to kill cancer cells,respectively [1,2].Recently,nanoparticles with PTT capabilities have attracted a great deal of attention in the photothermal treatment of tumor cells.A number of inorganic nanomaterials,such as Au nano-materials(including nanoshells,nanorods,nanocubes and nanoc-ages)[3e6],carbon nanomaterials[7e10],Pd nanosheets[11], copper sulfide[12],copper selenide[13]and W18O49nanoparticles [14]have also been shown to generate photothermal heating by NIR optical illumination to destroy cancer cells.The uses of conductive polymers-based nanomaterials[15e17]as PTT agents have also attracted significant attention.However,these nano-materials have very weak ability to generate reactive oxygen spe-cies(ROS)and are incapable for PDT application.Meanwhile,the most widely used commercial PDT agents are based on porphyrin, however,the maximal absorption wavelength of porphyrin is located in visible region,which is not suitable for in-depth tumor treatment through tissue.As a result,few examples of these PDT agents based on porphyrin have been applied to PTT[18,19]. Therefore,the combined therapy of PDT and PTT has rarely been developed to date.To generate thermal and ROS effectively for combination PTT and PDT,an ideal agent should exhibit strong absorbance band in the NIR region,which is a transparency window for biological tis-sues,and possess high photothermal conversion efficiency[17].To date,only two types of compounds,that is,NIR-absorbing BODIPY dyes and phthalocyanine(Pc)can meet the aforementioned de-mands.However,organic BODIPY dyes are limited by rapid pho-tobleaching.Phthalocyanine compounds,as one of the main class of photosensitizers,have been approved by the US Food and Drug Administration for clinical applications in the treatment of cancer due to their ability to generate singlet oxygen upon irradiation of light[20].Phthalocyanine derivatives have been extensively studied for their excellent stability against heat,light and harsh*Corresponding authors.Fax:þ862155664185.**Corresponding author.E-mail addresses:fengweifd@(W.Feng),wlya@(L.Y.Wang), fyli@(F.Y.Li).Contents lists available at ScienceDirectBiomaterialsjournal homepage:w ww.elsevi/locate/biomaterials0142-9612/$e see front matterÓ2013Elsevier Ltd.All rights reserved./10.1016/j.biomaterials.2013.07.027Biomaterials34(2013)7905e7912chemical environments[21].Although phthalocyanine shows considerable adsorption band at NIR region,few efforts have been devoted to investigate its application of phthalocyanine as PTT agent.To the best of our knowledge,the potential use of the phthalocyanine parent structure for PTT of cancer has not yet been reported due to its hydrophobicity.Our interest is developing a general administration route to fabricate nanocomposite and study the dual PTT and PDT effect, utilizing phthalocyanine as a model sensitizer.In this present study, hollow silica nanospheres(HSNs)with aqueous dispersibility and high stability were employed to load the hydrophobic phthalocy-anine(denoted as Pc@HSNs).Upon irradiated with NIR(730nm) laser,the Pc@HSNs can rapidly convert optical energy into heat and generate ROS after laser irradiation to eliminate tumors.The cor-responding PDT and PTT effects were evaluated in both cell level and animal models of tumor-bearing mice.The dual therapeutic properties from a single nanoparticle will play a significant role in promoting the use of hydrophobic photosensitizers in tumor therapies.2.Experimental section2.1.Materials and characterizationAll the starting materials were obtained from commercial supplies and used as received without further purification.Pluronic F108(EO132PO50EO132,where EO is polyethylene oxide and PO is polypropylene oxide)were purchased from Sigma e Aldrich.1,3,5-Trimethylbenzene,tetraethyl orthosilicate(TEOS),dimethyldime-thoxysilane(DMDMS),HCl,and AgNO3were purchased from Shanghai Chemical Corp.Phthalocyanine(Pc)was purchased from Sigma e Aldrich.Deionized water was used throughout the experiments.The morphology of HSNs was observed on a JEOL JEM-2010F transmission electron microscope(TEM)operated at200kV.Dynamic light scattering was carried out on an ALV-5000spectrometer-goniometer equipped with an ALV/LSE-5004light scattering electronic and multiple tau digital correlator and a JDS Uniphase He e Ne laser(632.8nm)with an output power of22mW.UV e vis spectra were measured with a UV e vis spectrophotometer(Agilent,8453).Nitrogen sorption isotherms were measured at77K with a Quantachrome Quardrasorb analyzer.Before mea-surement,the sample was degassed at180 C in vacuum for6h.The specific surface area was calculated using Brumauer e Emmet e Teller(BET)method,the pore size distribution was derived from the adsorption branches of the isotherms based on the Barrett e Joyner e Halenda(BJH)model.Weight changes of the products were monitored using a Mettler Toledo TGA-SDTA851analyzer(Switzerland)from25 C to750 C with a heating rate of10 C minÀ1.2.2.Synthesis of hollow silica nanospheres(HSNs)The HSNs were synthesized according to a previously reported procedure[22]. In a typical synthesis,1.0g of pluronic F108and1.0g of1,3,5-trimethylbenzene were added to30mL2.0M HCl and stirred vigorously for6h at25 C to form a homo-geneous emulsion.Then1.0g of TEOS was added to the surfactant solution under vigorous stirring.After6h of the reaction,0.5g of DMDMS was added and the re-action was continued for another48h.The milky mixture was dialyzed with a semipermeable membrane(molecular-weight cutoff,MWCO¼14,000)in500mL water for36h,and the water was refreshed every12h.The dialysate was evaporated at80 C,and the obtained white powder was calcined at350 C for5h to get the final product HSNs.2.3.The loading of phthalocyanine to HSNs0.1g of HSNs and0.1g of phthalocyanine were added to10mL chloroform.The mixture was stirred at40 C for12h and centrifuged(12,000rpm)to allow theHSNsFig.1.(a,b)TEM images of HSNs with different magnification.1a insert:the size distribution of HSNs.(c)DLS of HSNs samples disperse in water.(d)N2adsorption isotherm and pore size distribution curve of solid HSNs calculated from the adsorption branch by the NLDFT method before and after adsorption of phthalocyanine.J.Peng et al./Biomaterials34(2013)7905e79127906load phthalocyanine(called as Pc@HSNs).The obtained Pc@HSNs was washed with ethanol for several times until the supernatant was almost transparent after centrifugation to remove the unloaded phthalocyanine completely.Finally,the Pc@HSNs was dried at80 C.The loading ratio of the phthalocyanine inside HSNs was determined by UV-Vis absorption spectrum.2.4.Investigation of the release and photostability of Pc@HSNsTo investigate the possible release of phthalocyanine from Pc@HSNs in the physiological environment,2mg/mL Pc@HSNs was soaked in phosphate buffer so-lution(PBS)or RMPI1640nutrition medium for one week.Then the solution was centrifuged and the concentration of the phthalocyanine in the supernatant was analyzed using UV e vis spectroscopy.To investigate the photostability of Pc@HSNs, another group of2mg/mL Pc@HSNs was socked in PBS or RMPI1640nutrition medium for one week,irradiated by a730nm laser with a power of1.5W/cm2for 10min every day.The concentration of the phthalocyanine in the supernatant was monitored using UV e vis spectroscopy.2.5.Examination of photothermal effect for Pc@HSNs in aqueous solutionThe Pc@HSNs at the concentration of1mg/mL was irradiated using a730nm laser with a power density of1.5W/cm2.The temperature of solution was measured with infrared imaging devices(FLIR E40of FLIR Systems,Inc.,United States)at20ms intervals for a total of10min.Each solution was measured three times.2.6.ROS generation of Pc@HSNsThe generation of ROS was monitored by Image-iT LIVE Reactive Oxygen Species Kit(Molecular Probes/Beyotime)based on5-(and-6)-carboxy-20,70-dichlorodihy-drofluoresceindiacetate(carboxy-H2DCFDA),following the manufacturer’s protocol. H2DCFDA is afluorogenic marker for ROS,which permeates live cells and is deacetylated by intracellular esterases.In the presence of ROS,the reducedfluo-rescein compound is oxidized and emits bright greenfluorescence.Pre-seeded KB cells were incubated with Pc@HSNs suspension(250m g/mL)for3h at37 C with5% CO2.After washing the cells thoroughly with PBS,the cells were irradiated with a 730nm laser for3min H2DCFDA(10m M)was added and the cells were incubated at 37 C for30min.Fluorescence intensity was measured on an OLYMPUS FV1000 confocalfluorescence microscope.2.7.Cytotoxicity assessmentsCell-viability following PTT was determined by a methyl thiazolyl tetrazolium (MTT)assay.Briefly,cells were plated in96-wellflat-bottomed plates with a con-centration of5Â104cells per well and allowed to grow overnight prior to the exposure to HSNs or Pc@HSNs with different concentrations,and another group exposure to HSNs or Pc@HSNs with different concentrations for3h to allow the cell phagocytize nanoparticles,followed by irradiation with laser.After24h(including the phagocytosis time)of further incubation,MTT(20m L,5mg/mL)was added to each well and the plate was incubated for an additional4h at37 C under5%CO2to allow the conversion of MTT into a purple formazan product by active mitochondria. Then the formazan product was dissolved in dimethyl sulfoxide(DMSO)and quantified by the absorbance at570nm,with background subtraction at690nm, which was measured by means of a Tecan Infinite M200monochromator-based multifunction microplate reader.The following formula was used to calculate the inhibition of cell growth:Cell viability(%)¼(mean of Abs.value of treatment group/ mean Abs.value of control)Â100%.2.8.In vivo photothermal effect of Pc@HSNsThe S180mice sarcoma were inoculated to the male BALB/c mice(n¼4,6-week-old).When the tumor length reached50e70mm,the mice were intratumorally injected with Pc@HSNs suspension(100m L,2mg/mL).For control groups,mice were treated with the same volume of saline.Mice with and without Pc@HSNs injection were irradiated with the730nm laser(Hi-Tech Optoelectronics Co.,Ltd.Beijing,China) at power densities of1.5W/cm2for10min every day.The length(l)and width(w)of the tumors were measured by a vernier caliper,and the volume(V)of the tumor was estimated by the following formula:V¼4p/3Â(lw)2.Relative tumor volumes were calculated as V/V0(V0is the tumor volume when the treatment was initiated).2.9.Histological assessmentIn the test group,Kunming mice(n¼3)were intravenously injected with Pc@HSNs at a total dose of5mg/mL(0.4mL).And Kunming mice(n¼3)with no injection were selected as the control group.Blood samples and tissues were har-vested from test and control group after96h.Blood was collected from the orbital sinus by quickly removing the eyeball from the socket with a pair of tissue forceps. Upon completion of the blood collection,mice were sacrificed.The heart,liver,Fig.2.UV e vis spectra of Pc@HSNs solutions at a concentration of1mg/mL Inset:Photo of water(left)and Pc@HSNs(right)solutions at a concentration of1mg/mL in water.(b) Heating curves of water and Pc@HSNs(1mg/mL,2mL)under730nm laser irradiation at a power density of1.5W/cm2.(c)IR thermal images of water and Pc@HSNs solution exposed to the NIR laser at power densities of1.5W/cm2recorded at different time intervals,1:water;2:Pc@HSNs.J.Peng et al./Biomaterials34(2013)7905e79127907spleen,lung,and kidney were removed,and fixed in paraformaldehyde,embedded in paraf fin,sectioned,and stained with hematoxylin and eosin.3.Results and discussion3.1.Synthesis and characterization of Pc@HSNsHSNs were synthesized as carriers according to the method reported before [22].The as-prepared HSNs are hollow nano-spheres with uniform particle size,as shown in the transmission electron microscopy (TEM)image (Fig.1a).No apparent aggrega-tion of HSNs can be observed (Fig.1b).A statistic mean size of 35nm with a narrow size distribution (inset of Fig.1a)of HSNs is obtained by measuring the diameters of 100individual hollow nanospheres from the TEM image,demonstrating good mono-dispersity of the hollow nanospheres.Dynamic light scattering (DLS)measurements showed that HSNs exhibited a mean hydrated diameter of 37nm (Fig.1c),which agrees well with the statistical result observed from TEM.The nitrogen adsorption isotherm and the corresponding calculated pore size distribution curve of HSNsafter calcination at 350 C are shown in Fig.1d.A type IV adsorp-tion e desorption isotherm can be observed (Fig.1d).A uniform BJH pore size distribution centered at 24nm can be deduced for HSNs,which also in good accordance with the diameters of the cavity observed in the TEM image.The pore volume is 1.10cm 3/g,and the BET surface area is 579m 2/g.The wall thickness of the HSNs is calculated to be w 6.5nm.After loaded with phthalocyanine,the pore volume and surface area decrease signi ficantly to 0.28cm 3/g and 143m 2/g,respectively,proving that the phthalocyanine was adsorbed inside the hollow of the HSNs.The loading ratio of phthalocyanine in HSNs is measured to be 83.1%(W/W,Fig.S1),which is in accordance with thermal gravimetric analysis (TGA)results (Fig.S2).To testify the in vivo stability of Pc@HSNs,the material was soaked in PBS or RMPI1640nutrition medium for one week or with laser irradiation (730nm,1.5W/cm 2for 10min)every day.As shown in Fig.S3,even with 730nm irradiation,no obvious signal of phthalocyanine can be detected from the adsorption spectrum of the supernatants in both PBS and RPMI 1640,sug-gesting that the photosensitizer phthalocyanine was stably loaded inside the hollow cavities of HSNs withoutleakage.Fig.3.Confocal fluorescence images of KB cells to detect oxidative stress using Image-iT LIVE Reactive Oxygen Species (ROS)Kit.From top to down:untreated,irradiation with NIR (730nm,1W/cm 2,3min),treated with Pc@HSNs solution (250m g/mL),treated with Pc@HSNs solution (250mg/mL)followed by irradiation with NIR (730nm,1W/cm 2,3min).The cells showing green fluorescence color represent oxidatively stressed cells affected with ROS.FL images were collected at 510e 560nm,under excitation at 488nm.The scale bar is 50m m.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)J.Peng et al./Biomaterials 34(2013)7905e 79127908Fig.4.Confocalfluorescence images and brightfield images of KB cells.From top to bottom:untreated,irradiation with NIR(730nm,1.5W/cm2,10min),treated with Pc@HSNs solution(250m g/mL),treated with Pc@HSNs solution(250m g/mL)followed by irradiation with NIR(730nm,1.5W/cm2,10min).The cells are stained with PI,and the dead cells are observed in red.FL images were collected at600e680nm,under excitation at543nm.The scale bar is50m m.(For interpretation of the references to colour in thisfigure legend,thereader is referred to the web version of this article.)(b)Cells viabilities after phototherapy treatment under different Pc@HSNs concentration.Quantified KB cell viability from various groups.Data are expressed as meanÆs.d.(n¼3).3.2.Photothermal effect of Pc@HSNsPc@HSNs exhibited a high optical absorption coef ficient in the NIR range with a peak centered at w 760nm (Fig.2a).In order to verify the ef ficacy of Pc@HSNs as a photothermal agent,Pc@HSNs suspension with a concentration of 1mg/mL was exposed to 730nmlaser at a power density of 1.5W/cm 2.An obvious temperature in-crease was observed for Pc@HSNs suspension from 25 C to 50 C under laser irradiation (Fig.2b);the suspension was heated to over 45 C within 6min,which is capable for thermal treatment of tumor.While under the same conditions,pure water only showed a slight temperature raise of 3.5 C in 10min,demonstrating the raise of temperature is mainly contributed by Pc@HSNs rather than the ab-sorption of light by water.Following Roper ’s reported calculation method [23],the photothermal conversion ef ficiency (h )was calcu-lated to be 37.1%,using the following equation (1),the detailed cal-culations was supplied in Supporting Information .And Fig.2c shows the thermal images of Pc@HSNs suspension and pure water exposed to the 730nm laser recorded at different time intervals,where the difference in temperature can be visually observed.Xim i C p ;id Td t¼Q NP þQ DIS ÀQ Surr (1)where m and C p are the mass and heat capacity of water,respec-tively,T is the solution temperature,Q NP is the energy inputted by Pc@HSNs,Q Dis is the baseline energy inputted by the sample cell,and Q Surr is heat conduction away from the system surface by air.3.3.Intracellular ROS generated by Pc@HSNsAs designed,the Pc@HSNs will have PDT activity together with PTT ability upon 730nm illumination.The PDT activity to generate ROS of Pc@HSNs is evaluated by the detection of ROS generated within human epidermoid mouth carcinoma KB cells containing Pc@HSNs,upon irradiation with 730nm laser.The ROS production was assessed using the Image-iT LIVE Reactive Oxygen Species Kit.This assay is based on 5-(and-6)-carboxy-20,70-dichlorodihydro-fluoresceindiacetate (carboxy-H 2DCFDA)as a fluorogenicmarkerFig.6.IR thermal images of tumor-bearing mice with Pc@HSNs injection exposed to the NIR laser at power densities of 1.5W/cm 2recorded at different time intervals.As the control,IR thermal images of mice without Pc@HSNs injection exposed to the NIR laser at the power of 1.5W/cm 2weretaken.Fig.7.(a)Representative photos of mice bearing S180murine sarcoma after various different treatments indicated.The scale bar was 2cm.(b)Growth of S180tumors in different groups of mice after treatment.The relative tumor volumes were normalized to their initial sizes.Error bars were based on standard deviations.(c)Survival curves of mice after various treatments as indicated in (c).Pc@HSNs-injected mice after PTT treatment showed 100%survival ratio over 46days.J.Peng et al./Biomaterials 34(2013)7905e 79127910for ROS-permeated viable cells,and deacetylated by nonspecific intracellularesterase.In the presence of ROS,the reduced carboxy-H2DCFDA emits bright greenfluorescence when oxidized.There-fore,greenfluorescence can be observed from the cells oxidatively stressed by ROS.As shown in Fig.3,the test groups of untreated cells,laser-treated cells and Pc@HSNs-treated cells exhibited negligible production of ROS.However,Pc@HSNs-treated cells with 730nm irradiation showed substantially high greenfluorescence intensity.These results clarify that Pc@HSNs can generate ROS when excited with730nm light,therefore,Pc@HSNs can be applied for PDT therapy.3.4.In vitro cell viability assay of Pc@HSNsAs above-mentioned discussing,Pc@HSN has combination ability of both PTT and PDT effect.Thus,anti-cancer performance of Pc@HSNs in biological systems could be expected and was proved by in vitro cell experiments.KB cancer cells were incubated with Pc@HSNs at a concentration of250m g/mL for3h,and then irra-diated by the730nm laser with a power density of1.5W/cm2for 8min.After irradiation,the cells were stained by propidium iodide (PI)to indicate dead(red)cells,and imaged by a confocalfluores-cence microscope.As shown in Fig.4,significant cell death indi-cated by redfluorescence can only be observed in the test group of cells incubated with Pc@HSNs and followed by NIR irradiation.Standard cell viability tests were carried out to test the potential cell toxicity of Pc@HSNs.It was found that Pc@HSNs,even at a high concentration up to0.5mg/mL,exhibited no appreciable negative effect on the viability of cells after24h exposure(Fig.5a).MTT assay was also performed to quantitatively measure the relative cell via-bilities after phototherapy treatment under different concentrations of Pc@HSNs.As the concentration of Pc@HSNs was increased,more cells were killed by the laser irradiation(Fig.5b).The majority of cells were destroyed after being incubated with400m g/mL of Pc@HSNs and exposed to the730nm laser at1.5W/cm2for8min.In contrast, cells treated by only laser or HSNs with different concentration were not affected(Fig.S4).3.5.Photo-therapeutic efficacy of Pc@HSNs in vivoIn vivo phototherapy tests using Pc@HSNs as photosensitizer were performed on tumor-bearing mice.To monitor the photo-thermal effect in vivo of Pc@HSNs,the changing of temperature in the tumor region was recorded using a thermal imaging apparatus. After being intratumorally injected with Pc@HSNs suspension (100m L,2mg/mL)for10min,S180tumor-bearing mice were exposed to a730nm laser at a power density of1.5W/cm2.Under irradiation,the temperature of the tumor increased significantly from w30 C to w47 C within5min(Fig.6).In comparison,the temperature of tumor without Pc@HSNs injection had almost no changes under same730nm irradiation conditions.As tumor cells and tissue are sensitive to heat,these results proved the promise of phthalocyanine as a PTT agent.Although phthalocyanine has been used as PDT agents in a number of pathological indications[24,25], the potential use of phthalocyanine for photothermal treatment of cancer has not yet been reported.The phototherapeutic efficacy of Pc@HSNs was further investi-gated.A highly malignant murine tumor cell line S180was selected as the tumor model.After the length of tumors on the back of the mice reached approximately50e70mm,Pc@HSNs suspension (100m L,2mg/mL)was intratumorally injected.The tumors of each mouse in the treatment group were then exposed to a730nm laser at a power density of1.5W/cm2for10min every day.Three other groups including untreated mice(control,n¼4),mice exposed to the730nm laser(laser only,n¼4),and Pc@HSNs-injected mice without laser irradiation(Pc@HSNs,n¼4)were used as control groups.Tumor sizes were measured every2days after treatment.It is remarkable that for the mice group treated with both Pc@HSNs and730nm laser,the solid tumor shrinked gradually and was completely eradicated from the mice after5days of treatment (Fig.7b).In contrast,neither laser irradiation at the current power density nor only Pc@HSNs injection can affect the tumor growth (Fig.7a).Meanwhile,mice in the three control groups showed mean life spans of20e32days,by contrast,mice in the treated group(Pc@HSNsþlaser)were tumor-free after treatment,and survived over45days without a single death(Fig.7c).The above results proved that Pc@HSNs was a powerful agent for in vivo antitumor phototherapy.3.6.Histology and hematology results of the mice injected withPc@HSNsTo further evaluate the biosafety of Pc@HSNs,a serum biochem-istry assay and complete blood panel test were carried out using Pc@HSNs-injected(10mg/kg)healthy Balb/c mice at7days post in-jection.Notably,no detectable lesion(e.g.necrosis,hydropic degen-eration,inflammatory infiltrates,pulmonaryfibrosis,gastroenteritisFig.8.Histochemical study of the organs harvested from mice injected with Pc@HSNs (5mg/mL,0.4mL)for one week.The left and right rows are the control and experi-mental groups,respectively.J.Peng et al./Biomaterials34(2013)7905e79127911or infectious diarrhea)was observed for mice (Fig.8).Established serum biochemistry assays were also performed to further evaluate the toxicity to organs,especially the liver and kidney.No signi ficant fluctuations of the three important hepatic function indicators,alanine aminotransferase (ALT),aspartate aminotransferase (AST)and total bilirubin can be observed (Fig.9).The enzyme level of mice injected with Pc@HSNs was in the normal range.Two other indicators related to kidney function,creatinine and urea were also normal.The results collectively show that Pc@HSNs possess no noticeable toxic to mice in vivo .However,more effort is still required to systematically examine the potential long-term toxicity of our nanoparticles at various doses in animals.4.ConclusionWe demonstrated hollow silica nanoparticles loaded with hy-drophobic phthalocyanine Pc@HSNs for near-infrared photodynamic and photothermal combination therapy of cancer in vitro and in vivo .Using HSNs with good water dispersity and stability as host,Pc@HSNs are stable in physiological environments and nontoxic to cells at the tested concentrations.By intratumoral injection of Pc@HSNs,we further realized excellent tumor treatment ef ficacy using a 730nm laser irradiation at 1.5W/cm 2and achieved tumor elimination,without observing signi ficant toxic effects after treat-ment.Although the original phthalocyanine photosensitizer has been recognized only as a PDT agent,Pc@HSNs exhibit dual PDT and PTT properties,as both in vitro and in vivo phototherapeutic exper-iments against cancer cells demonstrated successful eradication of cancer cells.We believe that our results will open a new strategy for designing next-generation photosensitizers for cancer phototherapy.Con flict of interestNo financial con flict of interest was reported by the authors of this paper.AcknowledgmentsThis work was financially supported by NSFC (21231004and 21201038),MOST (2011AA03A407and 2013CB733700).Appendix A.Supplementary dataSupplementary data related to this article can be found at /10.1016/j.biomaterials.2013.07.027.References[1]Lal S,Clare SE,Halas NJ.Nanoshell-enabled photothermal cancer therapy:impending clinical impact.Acc Chem Res 2008;41:1842e 51.[2]Bardhan R,Lal S,Joshi A,Halas NJ.Theranostic nanoshells:from probe designto imaging and treatment of cancer.Acc 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简单的溶剂热法制备分层的碲化铋微米结构谭乃迪;张延林;陈峰;陈哲【摘要】采用简单的溶剂热法制备出高纯度的由纳米片自组装而形成的碲化铋微米结构.在碲化铋的形成中,乙二醇不仅作为溶剂,而且还作为还原剂.研究发现,聚乙烯吡咯烷酮(PVP)和硝酸在碲化铋的形成中起到了很重要的作用.通过X-射线衍射(XRD)、X-射线光电子能谱(XPS)、扫描电镜(SEM)、透射电镜(TEM)、高分辨透射电镜(HRTEM)、选区电子衍射(SAED)对其进行表征及研究.最后,利用时间演化实验对碲化铋的形成机理进行了探讨.%High-quality hierarchical microstructures of Bi2Te3 self-assembled by nanoplates were prepared via a simple wet chemical method under solvothermal conditions.Ethylene glycol was used not only as a solvent,but also as a reducing agent.The poly(vinyl pyrrolidone)(PVP) and nitric acid played an important role in the construction of the hierarchically self-assembled microstructures.The products were characterized by X-ray diffraction (XRD),X-ray photoelectron spectra (XPS),scanning electron microscopy (SEM),transmission electron microscopy (TEM),high-resolution transmission electron microscopy (HRTEM),and selected area electron diffraction (SAED). A formation mechanism was also proposed based on the result of time-dependent microstructures.【期刊名称】《无机化学学报》【年(卷),期】2012(028)010【总页数】7页(P2241-2247)【关键词】碲化铋;分层微米结构;聚乙烯吡咯烷酮;硝酸【作者】谭乃迪;张延林;陈峰;陈哲【作者单位】吉林化工学院,吉林132022;东北电力大学,吉林 132012;吉林石化公司有机合成厂,吉林 132021;吉林化工学院,吉林132022【正文语种】中文【中图分类】O612.5In the past few decades,synthesis of micro-and nano-scale inorganic materials have attracted efforts to develop the materials because of their outstanding properties and potential applications correlated with the morphology ofthe nanomaterials[1-6].Specifically,hierarchical architecture via self-assembly has become an intensive and hot research topic because of their unique physical and chemical properties and potential applications in optics,electronics,medicine,ceramics,catalysis,and energy/chemical conversions,and their important roles in the systematic research of structureproperty relationships in recently years[7-11].Thus,three-dimensional(3D)hierarchical architectures assembled by nanostructured building blocks such as nanoplates,nanoparticles, nanoribbons, and nanorods, were extensively investigated[12-16].Up to now,a wide variety of inorganic materials,including metal oxides,sulfides,hydrates,and other minerals,have been prepared with hierarchical structures.Forinstance,Zhu,et al.[17]reported the synthesis of hierarchical AlOOH nanostructured microspheresbymicrowave-assisted solvothermalrge-scale CeO2hierarchical architectures composed of well-aligned nanorods were prepared through hydrothermal method controllably by Chen and co-workers[18].Up to the present,much advancehasbeen made,butthefabrication of hierarchical 3D architecture is still a big challenge.The binary chalcogenides of AV2BVI3(A=As,Sb,Bi;B=S,Se,Te)are a class of important semiconductor materials having applications in thermoelectric,optoelectronic devices,IRspectroscopy,paints,photoemitting diodes,and microwave switches,etc[19-21].Bismuth telluride is one of the best thermoelectric materials because they are available for thermoelectric application near room temperature.They are widely used in thermopiles, thermal sensors, and thermoelectric cooler for laser diodes[22-24].In this regard,much progress has been made in the synthesis of Bi2Te3multifarious morphology,such as Bi2Te3intermetallic compounds with hierarchical nanostructures synthesized via an electrochemical deposition route[25],Bi2Te3plate-like crystals with homogeneous hexagonal morphology synthesized using a microwave assisted wet chemical method[26],and Bi2Te3nanoparticles and nanowiresprepared bysolvothermalmethod with NaBH4as thereductant[27].Great progress has been achieved in the synthesis approach,however,most of the above investigations also use additional inorganic reducing agents,for example,NaBH4,N2H4,and soon.Thusdeveloping convenientand fastersynthetic process using inexpensive,environmentally benignant reagents at lower temperature arestill important themes.Herein,we report a solvothermal process to prepare well-definedBi2Te3nanostructures selfassembled through nanoplates using Bi(NO)3and Te powder as precursors,and poly(vinyl pyrrolidone)(PVP)as a soft template in ethylene glycol(EG).All chemicals were used as receieved without furtherpurification.Bi(NO)3·5H2O and PVP(K90)were purchased from Sinopharm Chemical Reagent Co.Ltd.;Te powder was from Jingchun Chemical Reagents Company;Ethylene glycol was from Tianjin Guangfu Chemical Reagents Company;HNO3(85wt%solution)was from Beijing Chemical Reagents Company.Bi2Te3with different nanostructures were prepared by a simple solvothermal method in the presence of PVP.In a typical synthesis,3 mmol·L-1Bi(NO)3·5H2O and 9 mmol·L-1PVP (K90)were dissolved in 60 mL ethyle ne glycol.Then,4.5 mmol·L-1Te powder,and an appropriate quantity of HNO3aqueous solution(5 mol·L-1)were added into the above solution and transferred into an 80 mL Teflon-lined stainless steel autoclave under stirring.The autoclave was maintained at 180 ℃f or 48 h and then cooled to room temperature in air.The resulting solid product was collected and washed with deionized water and ethanol,and finally dried at room temperature for 12 h.The resulting samples were characterized by Rigaku D/Max2500PC X-ray po wder diffraction(XRD)equipped with graphitemonochromatizedCu Kα radiation(λ=0.154 18 nm)under the following conditions:30 kV asaccelerating voltage;100 mA as emission current;and the 2θ range of 5°～90°at a scan rate of 4°·min-1.The morphology and dimension of samples were observed by field emission scanning electron microscopy (FE-SEM,JEOL 7500B)and transmission electron microscopy (TEM,H-800).The microstructure of samples was determined using highresolution transmission electron microscopy(HRTEM)on a JEM-2010 apparatus with an acceleration voltage of 200 kV.X-ray photoelectonspectroscopy(XPS,ESCALAB 250)was used to confirm the oxidation stateof iron.A typical powder X-ray diffraction (XRD)pattern of the as-synthesized powder formed via templateassisted solvothermal synthesis is shown in Fig.1.All of the diffraction peaks can be readily indexed to pure rhombohedral Bi2Te3crystal(PDF No.15-0863).No impurities such asBi2O3and others,which often appear in the Bi2Te3product synthesized by traditional routes,are observed.Furthermore,all diffraction peaks are strong and narrow,indicating the high crystallinity of the Bi2Te3sample.The X-ray photoelectron spectroscopy(XPS)of the as-prepared sample obtained in the presence of PVP at 180℃was measured to examine the composition of the surface.The wide XPS spectrum in Fig.2a shows that no obvious impurities are detected in the XPS survey spectrum of Bi2Te3.It should be noted that only ethylene glycol solvent was used and no other reducing agent was introduced in our system.To prove whether or not ethylene glycol could completely reduce the Te powder to Te2-,XPS was also consulted to unambiguously assign the crystal phase.Fig.2b presentsthe XPS spectrum of Bi and Te element on the surface of the sample.The two strong peaks at 572.0 eV and 582.3 eV can be attributed to the bonding energies of Te3d5/2.The peaks at 575.7 eV and 582.3 eV can be assigned to the bonding energy of Bi3d3/2transition,which perfectly match with the previously published spectra of Bi2Te3[28].The composition of the formed Bi2Te3is determined by EDX analysis(Fig.2c)attached to a SEM instrument.The EDS analysis microanalysis clearly demonstrates that only Bi and Te element in the sample with an atomic ratio of Bi/Te is1∶1.51,which is consistent with stoichiometric ratio of Bi2Te3.No other impurities are detected in the EDX analysis,such as C,H,and O,indicating the complete disappearance of the surfactants (PVP)during the washing with deionized water and ethanol later process.Si and Au in the EDS spectrum (Fig.2c)originate from the silicon wafer to support and a thin Au layer sputtered on the sample,respectively.Thus,the XRD,XPS,and EDX spectrum analysis further confirm the high purity forBi2Te3sample,respectively.The morphology and size of the as-prepared Bi2Te3sample were characterized by FE-SEM and TEM.A panoramic SEM (Fig.3a)image demonstrates that the product displays sawtooth-like pattern.No other morphologies are observed,indicating a high yield of this sample.Asshown bythehighermagnification SEM image in Fig.3b,these sawtoothlike samples are composed of uniform nanoplates with diameters of 30～40 nm.The representative TEM images of the Bi2Te3samples obtained at 180℃are shown in Fig.3c and Fig.3d.As indicated by the figures,the as-preparedBi2Te3samples are consisted of sawtooth rods with diameters of 30～40 nm.Closer observations are shown in Fig.3d,the individual sawtooth rod has an average diameter of 35±10 nm and length of 1 μm,in good accordance with the SEM images.Further structural characterizations for the Bi2Te3sampleswerecarried outbySAED and HRTEM (Fig.3e).A selected area electron diffraction(SAED)pattern (inset in Fig.3e)taken from the edge of sawtooth rods can be indexed as a pure crystalline feature.Fig.3e shows a high-resolution TEM(HRTEM)image taken on individualnanoplate,revealing that each nanoplate is ploy-crystalline and the clear crystal lattices with d-spacing of 0.345 nm,which is nearly consistent with the(003)reection plane spacing of the rhombohedral Bi2Te3phase.Thus,all the abovementioned results clearly demonstrate the successful fabrication of the title material.The current synthesis strategy provides powerful means to tailor the composition,purity,and assembly of Bi2Te3microstructures as a function ofreaction parameters such as concentration of reagents,molar ratio of nitric acid(HNO3),and addition of PVP.Firstly,to study the influences of nitric acid on the morphology of as-synthesized Bi2Te3,controllable experiments were performed by changing reaction parameters.The nitric acid is essential for the development of the sawtooth rod.Fig.4 shows the TEM image of the sample synthesized in the same experimental condition except the volume of nitric acid.Perfect rose-like microflowers with diameters of 1 ～1.5 μm are formed witho ut the addition of nitricacid(Fig.4a).When 2 mL of the above nitric acid aqueous solution is used,itcan be clearly seen that the products are comprised of a large number of irregular and nonuniform plates with different sizes and a little of sawtooth rod,as shown in Fig.4b.With the the nitric acid volume added (3 mL)is increased,the perfect 3D flowerlike nanostructures with well-arranged nanoplates are obtained (Fig.4c).Secondly,Fig.5 shows the influence of PVP on the assembly behavior of Bi2Te3.The irregular and nonuniform nanoplates with a small size are formed without the used of PVP as shown in Fig.5a.When the PVP usage is increased to 3 mmol·L-1(Fig.5b),both irregular nanoplates and sawtooth rods are obtained.Fig.5c further exhibits the morphologies o f the products when 9 mmol·L-1PVP is used,namely the optimum synthesized condition,and a great deal of well-defined sawtooth-like nanostructures self-assembled by nanoplates are observed.Further increase in the concentration of PVP (27 mmol·L-1)leads to the formation of sawtooth-like microstructures with large length and width.Thus,these observations clearly show that the concentration of PVP in the system also has a great effect on the morphology of the products(Fig.5d).Finally,the concentration of reactants is another important influence factor on the morphology of Bi2Te3sample.Fig.6 presents TEM imagesofBi2Te3nanostructures obtained with different concentrations of reactants.When 5 mmol·L-1of Bi(NO3)3is used,as shown in Fig.6a,a few of nanorods with different diametersare found in the product.When the concentration of initial reagents is increased from 0.5 mmol·L-1to 1 mmol·L-1and other reaction parameters are similar to the product shown in Fig.6b,lots of welldefined dispersed sawtooth-like microstructures areobtained.If only 3 mmol·L-1of Bi(NO3)3is used,the resulting sample has irregular morphology(Fig.6c).According to the above results,we can conclude that the sawtooth-like microstructures strongly depend on the concentrations of Bi(NO3)3and PVP,molar ratio of nitric acid,respectively. To further confirm the formation mechanism of these sawtooth-like microstructures,time-dependent experiments were performed.With a short reaction time (12 h,Fig.7a),the resulted sample are consisted of a great deal of irregular nanoplates and minor amount of nanorods.When the reaction time is up to 24 h,the amount of sawtooth-like morphology are enhanced and nanoplates are decreased comparing with Fig.7a (see Fig.7b).With increasing the reaction time to 36 h,sawtooth rods with different sizes are increased (Fig.7c).It is worth pointing out that nanoplates are far smaller than the one prepared through 24 h.After 36 h of reaction time,nanoplates disappeare, and 3D hierarchical sawtooth-like microstructures with a diameter of 35 nm are obtained(Fig.7d).Although the exactmechanism forthe formation of the sawtooth rods microstructures now is not clear,on the basis of the experimental results,a possible interpretation for the formation process of the sawtooth-like structure could be given as follows:at the initial stage of reaction,Te powder may be dissolved in nitric acid[29],and Te2-is produced from the reduction of Te powder by the EG.Then,tiny Bi2Te3crystal nuclei are formed between Bi3+and Te2-through homogeneous nucleation.Further growth of thesecrystalnucleiresultsin the formation of nanorodsand nanoplates because oftheirhigh activity.The initially formed nanoplatesbegin to assemble in face-to-face growth style and nanorods are located at the center,owning to the interaction with PVP.With time goingon,perfect 3D hierarchical sawtooth-like microstructures are formed.In summary,high purity Bi2Te3material with hierarchical microstructure self-assembled by nanoplates has been synthesized in mild ethylene glycol hydrothermal system. 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