1、Mitochondria-Targeting Nanoplatform with Fluorescent Carbon Dotsfor Long Time Imaging and Magnetic Field-Enhanced Cellular UptakeYe Zhang,Yajing Shen,Xiyao Teng,Manqing Yan,Hong Bi,*,and Paulo Cesar Morais,College of Chemistry and Chemical Engineering, Anhui University, Hefei 230601, ChinaInstitute
2、of Health Sciences, Anhui University, Hefei 230601, ChinaSchool of Automation, Huazhong University of Science Technology, Wuhan 430074, ChinaInstituto de Fsica, Universidade de Braslia, Braslia, Federal District 70910-900, Brazil*S Supporting InformationABSTRACT: In this study, a biocompatible nanop
3、latform hasbeen constructed on the basis of magnetic mesoporous silicananoparticles (Fe3O4mSiO2) via surface modication oftriphenylphospine (TPP) and then conjugation with uores-cent carbon dots (CDs). The as-prepared Fe3O4mSiO2TPP/CDs nanoplatform shows a very low cytotoxicity andapoptosis rate in
4、various cell lines such as A549, CHO, HeLa,SH-SY5Y, HFF, and HMEC-1. More importantly, thisnanoplatform integrates long time cell imaging, mitochon-dria-targeting, and magnetic eld-enhanced cellular uptakefunctionalities into an all-in-one system. Time-dependentmitochondrial colocalization in all of
5、 the cell lines has beenproved by using confocal laser scanning microscopy and ow cytometry, while the multicolored uorescence of the Fe3O4mSiO2TPP/CDs could remain bright and stable after coincubation for 24 h. In addition, the cellular uptake eciency could beenhanced in a short time as a static ma
6、gnetic eld of 0.30 T was applied to the coincubation system of A549 and HFF cell lines.This bionanoplatform may have potential applications in targeted drug delivery for mitochondria diseases as well as early cancerdiagnosis and treatment.KEYWORDS: mitochondrial targeting, carbon dots, magnetic eld,
7、 cellular uptake, bioimaging, nanoplatformINTRODUCTIONThe development of therapeutic approaches for cancertreatment demanding higher ecacy has fostered the engineer-ing of multifunctional nanosystems. A biocompatible multi-functional nanosystem, usually called a bionanoplatform, aimsto construct new
8、 approaches to integrate cancer treatment,imaging, and drug delivery functionalities in an all-in-onesystem.1,2In the evolution of bionanoplatforms engineering,superparamagnetic Fe3O4nanoparticles (NPs) are probablyone of the most appealing candidates, given the success of T2asa contrast agent for m
9、agnetic resonance imaging (MRI), as wellas superparamagnetism for magnetic-targeted drug delivery andmagnetohyperthermia (MHT) therapy.3,4Conjugated withuorescent quantum dots or photosensitive reagents, theFe3O4NPs-based bionanoplatform can combine a dual imagingprobe for cancer5with magnetically g
10、uided drug delivery,6targeted chemotherapy,7or photodynamic therapy (PDT),8and photothermal therapy.9In the past decades, mesoporoussilica nanoparticles (MSNs) are known to have little or notoxicity due to favorable biocompatibility of silicon. Moreover,surface modied or end-capped MSNs are excellen
11、t nanopalt-forms for targeting drug delivery and chemotherapy, owing totheir distinctive mesoporous structure, large surface area,tunable pore size, facile functionalization chemistry, and goodbiocompatibility.1012Particularly, the engineered design of amesoporous silica shell on a magnetite nanocry
12、stal core(Fe3O4mSiO2) endows the as-constructed nanoplatformwith multifunctionalities including MRI, uorescence imaging,and magnetic targeted-drug delivery.13In addition, magneticNPs under an applied magnetic eld could direct cancer-targeting drug delivery,14enhance T cell activation so as tostimula
13、te antitumor activity,15promote cell sorting,16andmodulate cellular uptake.17Though the mechanism and exactprocess of magnetic NPs cellular uptake assisted by an appliedmagnetic eld is still unclear, most of the research regarding themultifunctional Fe3O4mSiO2NPs has focused on combiningdrug deliver
14、y system (DDS) with biosensing18or MRIfunctionalities.19Recently, advances in biomedical nanotechnology makesubcellular-targeted therapy an emerging important area forcancer treatment. Compared to the random interaction ofdrugs with intracellular site of action, the therapeutic outcomesReceived: Jan
15、uary 14, 2015Accepted: April 27, 2015Research Articlewww.acsami.org XXXX American Chemical Society A DOI: 10.1021/acsami.5b00405ACS Appl. Mater. Interfaces XXXX, XXX, XXXXXXcould be enhanced manyfold if pharmaceutical agents can bespecically directed toward the targeted organelle, i.e., pro-apoptoti
16、c compounds to mitochondria.20,21In general, theadvantages of organelle targeting construction in chemotherapyare to achieve a higher drug ecacy as well as a minimum sideeect. The mitochondrion is a double-membrane of phospho-lipid bilayer enveloped cytoplasmic organelle that possesses itsown intern
17、al genome. The mitochondria are indispensable forproviding energy for the survival of eukaryotic cells and tocontrol the activation of programmed cell death (so-calledapoptosis) mechanism by regulating the translocation of pro-apoptotic proteins from the mitochondrial intermembranespace to the cytos
18、ol.22Therefore, the mitochondrion isrecognized as an important therapeutic target in cancer therapy,and many researchers have attempted to design mitochondria-targeted pharmaceuticals and drug carriers.2325Triphenyl-phosphonium (TPP) is a representative lipophilic cationicspecies that can selectivel
19、y accumulate in the mitochondria byreducing the free energy change while moving from an aqueousto the hydrophobic environment in response to themitochondrial membrane potential of about 180 to 200mV.26Through conjugation with TPP, several biologicallyactive molecules, such as coenzyme Q or vitamin E
20、 derivative,have been selectively targeted to mitochondria to enhanceantioxidant ecacy.27,28In recent years, various kinds ofmitochondria-targeted TPP-conjugated nanosystems have beendeveloped, including liposomes,29dendrimers,30polymericnanoparticles3134and small molecules.35Compared to thenontarge
21、ted counterparts, a TPP-conjugated nanosystem showsa remarkable improvement in the drug therapeutic index forcancer, Alzheimers disease, and obesity.31From last century, organic dyes and uorescent proteins havebeen widely used in many biomedical elds, but their poorphotobleaching property is not sui
22、table for long-term and real-time bioimaging. Fluorescent quantum dots (QDs) are newlyappealing high quality bioprobes for applications in diversebiological research because of their size-tunable emission,strong uorescence, and high photostability.36However,uorescent II/VI semiconductor QDs, one kin
23、d of the well-studied QDs, are handicapped by a potential toxicity problemdue to release of heavy metal ions (e.g., Cd ions).37Toovercome these limits in biomedical applications, biocompat-ible QDs, i.e., carbon QDs,38silicon QDs,39and novel nitrogen-rich QDs,40have attracted more and more attention
24、 and beingcontinuously studied. Among them, carbon quantum dots(CDs), as a new carbon material of less than 10 nm in size,41have been receiving growing research interest since they werediscovered in 2004.42CDs possess many advantages, such asease of production, high uorescent activity, resistance to
25、photobleaching, and excellent biocompatibility, make them verypromising for long time bioimaging.43,44Furthermore, it ispostulated that the doping of nitrogen atoms can introduce theCDs a new kind of surface state, and their quantum yields aregreatly elevated. Nitrogen and sulfur codoped CDs have be
26、ensynthesized from a precursor comprising L-cysteine and citricacid, showing high yield and excitation-independent emission.45Recently, the one-pot hydrothermal synthesis of nitrogen-doped CDs (NCDs) from dierent nitrogen sources has beenreported, and the obtained NCDs emit bright blueuorescence. Ce
27、llular toxicity test and bioimaging experimenthave demonstrated that NCDs not only possess low toxicity tocells but also have stronger resistance to photobleaching thanCDs and thus have better performance in labeling than CDs.46Previously, NCDs in size of 23 nm with a high quantum yield(22% in ethan
28、ol) as well as low cytotoxicity have beensynthesized and employed for live cell imaging in our lab.47Inthis paper, the as-synthesized CDs were conjugated with TPP-modied Fe3O4mSiO2to construct a novel biocompatiblenanoplatform (Fe3O4mSiO2TPP/CDs) for mitochondria-targeting, long time cell imaging, a
29、nd magnetic eld-enhancedcellular uptake.MATERIALS AND METHODSMaterials. Ferrous ammonium sulfate (FeSO4(NH4)2SO46H2O),sodium hydroxide (NaOH), oleic acid, cetyltrimethylammoniumbromide (CTAB), tetraethyl orthosilicate (TEOS), ethyl acetate(EtOAc), ammounium nitrate (NH4NO3), TPP, -bromobutyric acid,
30、thionyl chloride, n-hexane, chloroform, ethanol, ethylenediaminetetra-acetic acid (EDTA), and dimethyl sulfoxide (DMSO) were purchasedfrom Sinopharm Chemical Reagent Co. and used without furtherpurication. Dimethylformamide (DMF) and toluene were obtainedfrom Sinopharm Chemical Reagent Co. and disti
31、lled under reducedpressure before use. Albumin from bovine serum (BSA) was purchasedfrom Aladdin Industrial Corporation. 3-Aminopropyltriethoxysilane(APTES), uorescein isothiocyanate (FITC) and 3-(4,5-dimethylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchasedfrom Sigma-Aldrich Chemi
32、cals and used as received. The food-gradekonjac our (KF, 50 wt %) was donated from Biosharp Co. USA.Dulbeccos modied Eagles medium (DMEM), penicillin/streptomy-cin, and fetal bovine serum (FBS) were purchased from Hyclone. TheLDH assay kit was supplied by Nanjing Jian Chen BioChem Co.MCDB 131 medium
33、, microvascular growth supplement (MVGS),Mitotracker Red, and Lysotracker Red DND-99 were obtained fromInvitrogen. Hoechst 33258 and 4,6-diamidino-2-phenylindole (DAPI)were supplied by Beyotime Institute of Biotechnology. Ultrapure waterwas used throughout for all experiments.Synthesis of Fe3O4mSiO2
34、. First, uniform magnetite NPs wereprepared according to a typical liquidsolidsolid synthetic route asfollows.48The Fe2+-rich suspension was obtained by adding theFeSO4(NH4)2SO46H2O aqueous solution (0.15 M) into a mixturecomprising NaOH (2.0 g), oleic acid (20 mL), and ethanol (20 mL)followed by st
35、irring at room temperature for a period of time. Second,the prepared suspension was transferred to a 100 mL Teon-linedautoclave and maintained at 180 C for 10 h. The product wasextracted with n-hexane and ethanol and further puried by magneticseparation and washing with ethanol several times. Third,
36、 the obtainedoleic acid-capped Fe3O4NPs were dispersed in n-hexane again, andthen they were transferred from n-hexane to water in a typicalprocedure by adding CTAB aqueous solution (0.55 M).49The Fe3O4/CTAB aqueous solution was diluted into certain amount of water andthe temperature of the solution
37、was kept at 60 C. The NaOHsolution (2 M) was added to adjust the pH value to 12 and appropriateamounts of TEOS and EtOAc were slowly added into the reactionmedium in sequence. After speedy-stirring for 30 s, the suspension wasaged for 3 h. The Fe3O4mSiO2NPs were collected by centrifugationand washed
38、 with ethanol three times. To remove the excess of CTABfrom the product, the Fe3O4mSiO2NPs were dispersed into 10 mg/mL of NH4NO3/ethanol solution and reuxed twice at 80 C for 3 h.Synthesis of Fe3O4mSiO2TPP. As shown in Scheme S1 ofSupporting Information, at the beginning, amino-modied NPs(Fe3O4mSiO
39、2NH2) were prepared by suspending 0.10 g of theFe3O4mSiO2NPs in a round-bottom ask with 1 mL of APTES and50 mL of toluene and then reuxing for 24 h. The Fe3O4mSiO2NH2was then collected by centrifugation, washed with toluene andethanol, and dried under vacuum at 60 C. Subsequently,(carboxyethyl)triph
40、enylphosphonium bromide (CTPB) was obtainedas follows. 2.63 g of TPP was dissolved into 30 mL of toluene andheated up to 40 C. Once the temperature of the medium reached 40C, 0.20 g of -bromobutyric acid was quickly added and thesuspension was heated up to 120 C. The reaction medium was keptunder st
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