Intracellular Self-assembly of TPE-biotin Nanoparticles Enables Aggregation-Induced Emission Fluorescence for Cancer-Targeted Imaging

Yi-fei Xu,Jin-hui Jiang

a.Department of Nuclear Medicine,The First Affiliated Hospital of Anhui Medical University,Hefei 230022,China

b.Department of Molecular and Cell Biology,University of Leicester,Leicester,LE1 7RH,UK

Fluorogens with aggregation-induced emission(AIE)characteristics have recently been widely applied for studying biological events,and fluorogens with “smart” properties are especially desirable.Herein,we rationally designed and synthesized a biotinylated and reduction-activatable probe(Cys(StBu)-Lys(biotin)-Lys(TPE)-CBT(1))with AIE properties for cancer-targeted imaging.The biotinylated probe 1 can be actively uptaken by the biotin receptor-overexpressing cancer cells,and then “smartly” self-assemble into nanoparticles inside cells and turn the fluorescence “On”.Employing this “smart” strategy,we successfully applied probe 1 for cancer-targeted imaging.We envision that this biotinylated intelligent probe 1 might be further developed for cancer-targeted imaging in routine clinical studies in the near future.

Key words:Tetraphenylethylene,Biotin,Self-assembly,Aggregation-induced emission

I.INTRODUCTION

Aggregation-induced emission(AIE)fluorogens,a class of luminogenic materials,have recently been widely applied for biosensing and bioimaging due to their unique photophysical and optoelectronic properties[1,2]. As compared to conventional fluorophores that are limited by fluorescence quenching at high concentrations or in the aggregated state due to π-π stacking interactions,AIE fluorogens,such as tetraphenylethylene(TPE),are non-emissive in the dissolved state,but exhibit increased emission when aggregated[3].The AIE eflect results in excellent performance in imaging with a high signal-to-noise ratio,facile operation in living organisms,low cytotoxicity,few false-positive signals,and strong photobleaching resistance[4,5].To date,a variety of AIE-basedfluorescent probes have been developed for cell apoptosis[6],mitochondria[7],nuclease[8],and alkaline phosphatase[9].

Cancer cells overexpress many tumour-specific receptors which can be used as biomarkers to deliver imaging agents or drugs into tumours.Some ligands,such as monoclonal antibodies[10],folic acid[11],aptamers[12],oligopeptides[13],transferrin[14],and biotin[15],have been widely used as tumour-specific agents to design tumour-targeted drugs or fluorophore conjugates.Biotin,also known as vitamin B7,is a growth promoter at the cellular level and plays an important role in sustaining cell proliferation and growth[16].It has been reported that the biotin receptors are overexpressed in various cancer cell types[17].Accordingly,biotin appears to be well-suited in the development of cancertargeted techniques.

Click chemistry is a new type of chemical reaction with high efficiency and selectivity,which plays a key role in the development of molecular diversity and new technology in the field of biomedicine[18].Recently,during a study of fluorescein regeneration in fireflies,Rao and Liang[19]discovered a biocompatible click reaction between the cyano group of 2-cyanobenzothiazole(CBT)and the 1,2-aminothiol group of cysteine(Cys),which can be stimulated by protease,reduction or pH for the self-assembly of nanostructures.This click reaction has been widely used in intracellular hydrogelation[20],detection of intracellular biomolecules[21],and drug delivery systems[22].

Inspired by the above pioneering studies,as shown in FIG.1,we designed a biotinylated and reductionactivatable probe(Cys(StBu)-Lys(biotin)-Lys(TPE)-CBT(1)),the fluorescence of which was turned “On”upon reduction-controlled condensation and the AIE effect in targeted cancer cells for highly sensitive cancertargeted imaging.When probe 1 was subjected to glutathione reduction and self-assembled into TPE-biotin nanoparticles(i.e.,1-NPs),the AIE fluorescence was greatly enhanced,with which we successfully applied probe 1 for specific and highly sensitive imaging of biotin receptor-overexpressing HepG2 cancer cells.

II.EXPERIMENTS

A.Materials

All of the starting materials were purchased from Adamas or BaoMan Inc.(Shanghai,China).Commercially available reagents were used without further purification,unless noted otherwise.All chemicals were reagent grade or better.

B.General methods

High-performance liquid chromatography(HPLC)purification was performed on an Shimazu UFLC system equipped with two LC-20AP pumps and a SPD-20A UV/Vis detector using a Shimazu PRC-ODS column.HPLC analyses were performed on an Agilent 1200 system equipped with a G1322A pump and an in-line diode array UV detector on an Agilent Zorbax 300SD-C18RP column,with CH3CN(0.1%of TFA)and water(0.1%of TFA)as the eluent. Matrixassisted laser desorption(MALDI)ionization-time offlight(TOF)/TOF and ESI mass spectra were obtained on a time-of-flight Ultraflex II mass spectrometer(Bruker Daltonics)and a Finnigan LCQ Advantage ion trap mass spectrometer(Thermo Fisher Corporation)equipped with a standard ESI source,respectively.Transmission electron micrograph(TEM)images were obtained on a JEM-2100F field emission transmission electron microscope operated at an acceleration voltage of 200 kV.Cell images were obtained on an IX71fluorescence microscope(Olympus,Japan).Fluorescence spectra were recorded on an F-4600 fluorescence spectrophotometer(Hitachi High-Technologies Corporation,Japan)with the excitation wavelength set to 320 nm.

C.Syntheses of compounds

2-Cyano-6-aminobenzo-thiazole(CBT)was synthesized according to the literature[23].The synthetic route for 1 is shown in FIG.2(A).

1.Synthesis of compound B

Compound Fmoc-Cys(StBu)-Lys(biotin)-Lys(boc)-OH(A)was synthesized with solid phase peptide synthesis(SPPS).Isobutyl chloroformate(IBCF,109 mg(0.80 mmol))was added to a mixture of compound A(400 mg(0.40 mmol))and 4-methylmorpholine(MMP,81 mg(0.80 mmol))in THF(5.00 mL)at 0?C under N2.The reaction mixture was stirred for 1 h.The 2-cyano-6-aminobenzothiazole(CBT,71 mg(0.40 mmol))was added to the reaction mixture and further stirred at 0?C for 1 h. Then,the mixture was stirred overnight at room temperature.Compound B(281 mg,yield of 60%)was purified with HPLC using wateracetonitrile with 0.1%TFA as the eluent.MS calculated for B((M+H)+):1171.4601,observed HR-ESI/MS:m/z=1171.4637.

2.Synthesis of compound C

The Boc protecting groups of compound B were removed with dichloromethane(DCM,300μL)and triisopropylsilane(TIPS,200μL)in TFA(9.5 mL)for 3 h.Compound C(267 mg,yield of 95%)was obtained after HPLC purification using water-acetonitrile with 0.1%TFA as the eluent.MS calculated for compound C((M+H)+):1071.4077,observed HR-ESI/MS:m/z=1071.5296(FIG.2(B)).

3.Synthesis of compound D

TPE-COOH (150 mg, 0.40 mmol), N-hydroxysuccinimide(56 mg,0.48 mmol),and EDC·HCl(92 mg,0.48 mmol)were dissolved in 1 mL of DMF and stirred overnight at room temperature(RT)to yield TPE-ester,then compound C(214 mg,0.40 mmol)andN,N-diisopropylethylamine(DIPEA,78.1mg(0.57 mmol))was added to the mixture and further stirred for 12 h at RT to yield D.Compound D(406 mg,yield of 71%)was purified with HPLC using water-acetonitrile with 0.1%TFA as the eluent.MS calculatedforcompoundD ((M+H)+): 1429.54;observed ESI/MS:m/z=1429.72.

4.Synthesis of 1

The Fmoc protecting group of compound D was cleaved with 5%piperidine in DMF(10 mL)at 0?C for10min,then0.6mL ofTFA was added to neutralize the alkaline,yielding compound 1(272 mg,yield of 67%)after HPLC purification with water-acetonitrile as the eluent.MS calculated for 1((M+H)+):1208.0500,observed HR-MALDITOF/MS:m/z=1207.4754(FIG.2(B))).

III.RESULTS AND DISCUSSION

A.Rationale of the design

FIG.3(A)Fluorescence spectra of 5 μmol/L of 1(black)incubated with 50 μmol/L glutathione at 37 ?C for 1 h in PBS bufler(red).Excitation:320 nm.(B)TEM image of 1-NPs dispersion.(C)HPLC traces of 5μmol/L of 1(black)and 5 μmol/L of 1 incubated with 50 μmol/L glutathione at 37 ?C for 1 h(i.e.,1-NPs dispersion)(red)in PBS bufler.(D)HR-MALDI-TOF/MS spectrum of 1-dimer in FIG.3(C).

Our probe was designed with the following components,as shown in FIG.1(A):a disulfided cysteine(Cys)motif and a CBT motif for the CBT-Cys click condensation reaction,a biotin motif conjugated to the side chain of a lysine(Lys)motif,and a TPE motif conjugated to the side chain of a lysine(Lys)motif to generate an AIEfluorescence signal.At a neutral pH and in the presence of reducing agents(e.g.,glutathione(GSH)),a free 1,2-aminothiol group was generated and immediately condensed with the cyano group of the CBT motif to yield oligomers(most of which are dimers).Subsequently,the oligomers self-assemble into nanoparticles(1-NPs),as previously demonstrated[19–22].As illustrated in FIG.1(B),after being internalized by the biotin receptoroverexpressing cancer cells,probe 1 will be reduced to yield the active intermediate,1-Red,thus exposing a free 1,2-aminothiol group on the Cys motif.Then,the click reaction among the molecules of 1-Red immediately occurs to yield oligomers which self-assemble into 1-NPs,accompanied with an enhanced FL signal.By this means,the biotin receptor-overexpressing cancer cells are highly specific and highly sensitive imaged by the AIE probe.

B.In vitro AIE fluorescence detection of GSH with 1

After the pure compound 1 was fully characterized,we used GSH to trigger the condensation of 1 to self-assemble into TPE-biotin nanoparticles(1-NPs)in vitro and studied the AIE properties. In detail,5μmol/L of 1 in the phosphate-buflered saline(PBS)containing 10%DMSO(v/v,pH=7.4)was incubated with 50-fold GSH at 37?C for 1 h to prepare 1-NPs dispersion.As shown in FIG.3(A),compared to the fluorescence spectrum of 1,we noticed that the fluorescence of 1-NPs dispersion was turned “On” and the emission intensity at 470 nm increased approximately 6.2-fold,suggesting efficient AIE fluorescence turned “On” after nanoparticle formation.The TEM images clearly show the formation of 1-NPs(FIG.3(B)).Additionally,HPLC and HR-MALDI-TOF/MS analyses clearly indicate 1-NPs are composed of the condensation product of compound 1(i.e.,1-dimer,FIG.3(C)and(D)).

C.Cancer cell-targeted imaging assay

After confirming 1 could be efficiently reduced by GSH to turn the fluorescence “On”,which was then applied for cancer cell-targeted imaging.Both biothiol and biotin receptor-overexpressing hepatocellular carcinoma HepG2 cells were selected for this purpose[24].Before that,cytotoxicity of compound 1 on HepG2 cells was investigated and the results indicated that up to 12 h at 40μmol/L did not induce obvious cytotoxicity on HepG2 cells(FIG.4),suggesting that 5μmol/L of 1 is safe for cell imaging.Then,HepG2 cells were incubated with 5 μmol/L of 1 at 37 and 4?C for time course analyses.As shown in FIG.5(A),fluorescence of the HepG2 cells at 37?C gradually turned “On” and reached its intensity plateau after 2 h,suggesting efficiently cell uptake and fast intracellular condensation of 1.We thus chose an incubation time of 2 h for the following HepG2 cell-targeted imaging studies.After the HepG2 cells were incubated with 5 μmol/L 1 at 37?C for 2 h,as shown in the top row of FIG.6,bright fluorescence was observed from the cytoplasm,suggesting that probe 1 was reduced by biothiol in the lysosomal compartment and self-assembled,which induced an enhanced AIE fluorescence generation in the cells.

FIG.4 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay of 1 on HepG2 cells.Cell viability values(%)were estimated by MTT proliferation test with concentrations of 5,10,20,or 40μmol/L of 1.HepG2 cells were cultured in the presence of 1 for 3,6,or 12 h at 37?C under 5%CO2.These experiments were performed in triplicate.The results are representative of three independent experiments.Error bars represent standard deviations.

To verify the targeting eflect of biotin on the molecular probe 1,we conducted fluorescence imaging of HepG2 cells incubated with 1 at 4?C.As shown in FIG.5,at the same incubation time,the fluorescence inside the cells at 4?C was obviously lower than cells at 37?C.Quantitative analysis indicated that,as shown in FIG.5(B),within the observation time of 0.5?3 h,thefluorescence of the cells at 37?C increased much quicker than cells at 4?C.Moreover,we also pre-treated HepG2 cells with an excess of biotin for 2 h to block the biotin receptors and prevent or reduce competitive binding of probe 1.As shown in the bottom row of FIG.6,in agreement with expectations,HepG2 cells incubated with 5μmol/L of 1 at 37?C for 2 h showed reduced fluorescence.Those results were thus fully consistent with our hypothesis that probe 1 could actively target the biotin receptor-positive tumor cells via receptor-mediated endocytosis[25].This finding also proved that the biotin receptor on cell membranes is a good biomarker for cancer cell-targeted imaging.

IV.CONCLUSION

In summary,by employing the AIE properties of a TPE fluorogen and a biocompatible click condensationing. In vitro results indicated that when compared with probe 1,the formation of a TPE-biotin nanoparticle resulted in a 6.2-fold increase in fluorescence intensity.Cell experiments indicated that this type of biotinylated molecular probe could be actively taken up by biotin receptor-overexpressing tumor cells via receptor-mediated endocytosis,and intracellular selfassembly of the TPE-biotin nanoparticle results in enhanced AIE fluorescence.With the fluorescence“Turn-On”property,probe 1 was successfully applied for cancer-targeted imaging.On this basis,we conclude that our “smart” strategy of reduction-instructed AIE represents a potentially useful new approach to imaging design,and can be widely applied for cancer celltargeted imaging with dramatically enhanced sensitivity.

FIG.5(A)Top row:fluorescence-microscopic images of HepG2 cells incubated with 5μmol/L of 1 in culture medium containing 2%DMSO at 37?C for 0.5,1,1.5,2,2.5,and 3 h.Bottom row:fluorescence-microscopic images of HepG2 cells incubated with 5μmol/L of 1 in culture medium containing 2%DMSO at 4?C for 0.5,1,1.5,2,2.5,and 3 h.All images have the same scale bar:10μm.(B)Quantification of the mean flux(photon/s)for the cells images in(A).37 ?C(black),4 ?C(red).

FIG.6 Top row:diflerential interference contrast image,fl uorescence and overlay images of biotin receptor-positive HepG2 cells after incubation with 5 μmol/L of 1 at 37 ?C for 2 h.Bottom row:diflerential interference contrast image,fluorescence and overlay images of the biotin receptorpositive HepG2 cells pretreated with 0.5 mmol/L of biotin at 37 ?C for 1 h,followed by incubation with 5μmol/L of 1 for 2 h,washed with PBS for three times prior to imaging,respectively.All images have the same scale bar:10μm.

V.ACKNOWLEDGEMENTS

This work was supported by Anhui Scientific and Technological Project(No.1704a0802164)and the Natural Science Foundation of the Anhui Higher Education Institutions of China(No.KJ2018A0192).

[1]G.Feng,R.T.Kwok,B.Z.Tang,and B.Liu,Appl.Phys.Rev.4,021307(2017).

[2]R.T.Kwok,C.W.Leung,J.W.Lam,and B.Z.Tang,Chem.Soc.Rev.44,4228(2015).

[3]J.Mei,Y.Hong,J.W.Lam,A.Qin,Y.Tang,and B.Z.Tang,Adv.Mater.26,5429(2014).

[4]J.Liang,B.Z.Tang,and B.Liu,Chem.Soc.Rev.44,2798(2015).

[5]Y.Yuan,C.J.Zhang,M.Gao,R.Zhang,B.Z.Tang,and B.Liu,Angew.Chem.Int.Ed.54,1780(2015).

[6]X.Gu,R.T.Kwok,J.W.Lam,and B.Z.Tang,Biomaterials 146,115(2017).

[7]X.Li,M.Jiang,J.W.Lam,B.Z.Tang,and J.Y.Qu,J.Am.Chem.Soc.139,17022(2017).

[8]M.Wang,D.Zhang,G.Zhang,Y.Tang,S.Wang,and D.Zhu,Anal.Chem.80,6443(2008).

[9]M.C.Zhao,M.Wang,H.Liu,D.S.Liu,G.X.Zhang,D.Q.Zhang,and D.B.Zhu,Langmuir 25,676(2009).

[10]I.Ojima,Acc.Chem.Res.41,108(2008).

[11]H.Wang,L.Zheng,C.Peng,M.Shen,X.Shi,and G.Zhang,Biomaterials 34,470(2013).

[12]H.M.So,K.Won,Y.H.Kim,B.K.Kim,B.H.Ryu,P.S.Na,H.Kim,and J.O.Lee,J.Am.Chem.Soc.127,11906(2005).

[13]Y.Du,S.Guo,H.Qin,S.Dong,and E.Wang,Chem.Commun.48,799(2012).

[14]S.Dixit,T.Novak,K.Miller,Y.Zhu,M.E.Kenney,and A.M.Broome,Nanoscale 7,1782(2015).

[15]J.H.Jiang,Z.B.Zhao,Z.J.Hai,H.Y.Wang,and G.L.Liang,Anal.Chem.89,9625(2017).

[16]D.N.Heo,D.H.Yang,H.J.Moon,J.B.Lee,M.S.Bae,S.C.Lee,W.J.Lee,I.C.Sun,and I.K.Kwon,Biomaterials.33,856(2012).

[17]S.Bhuniya,S.Maiti,E.J.Kim,H.Lee,J.L.Sessler,K.S.Hong,and J.S.Kim,Angew.Chem.Int.Ed.53,4469(2014).

[18]P.Thirumurugan,D.Matosiuk,and K.Jozwiak,Chem.Rev.113,4905(2013).

[19]G.Liang,H.Ren,and J.Rao,Nat.Chem.2,54(2010).

[20]Z.Zheng,P.Y.Chen,M.L.Xie,C.F.Wu,Y.F.Luo,W.T.Wang,J.Jiang,and G.L.Liang,J.Am.Chem.Soc.138,11128(2016).

[21]Z.J.Hai,J.J.Wu,D.Saimi,Y.H.Ni,R.B.Zhou,and G.L.Liang,Anal.Chem.90,1520(2018).

[22]Y.Yuan,L.Wang,W.Du,Z.L.Ding,J.Zhang,T.Han,L.N.An,H.F.Zhang,and G.L.Liang,Angew.Chem.Int.Ed.54,9700(2015).

[23]E.H.White,H.Worther,H.H.Seliger,and W.D.McElroy,J.Am.Chem.Soc.88,2015(1966).

[24]W.Young′aKim and J.Seung′aKim,Chem.Commun.51,9343(2015).

[25]N.Jiang,N.S.Tan,B.Ho,and J.L.Ding,Nat.Immunol 8,1114(2007).