AIEgen-Conjugated Magnetic Nanoparticles as MagneticâFluorescent

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AIEgen-Conjugated Magnetic Nanoparticles as Magnetic-Fluorescent Bioimaging Probes Kuheli Mandal, Debabrata Jana, Binay Krishna Ghorai, and Nikhil R. Jana ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00636 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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ACS Applied Nano Materials

AIEgen-Conjugated Magnetic Nanoparticles as MagneticFluorescent Bioimaging Probes Kuheli Mandal,1 Debabrata Jana,2 Binay K. Ghorai,2,* and Nikhil R. Jana 1,* 1Centre

for Advanced Materials and School of Materials Science, Indian Association for the Cultivation of Science, Kolkata 700 032, India

2Department

of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah 711 103, India

*Address correspondence to [email protected], [email protected]

1 ACS Paragon Plus Environment

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Abstract: Colloidal nanoparticle with magnetic-fluorescent property and varied surface chemistry has enormous biomedical application potential. However, the synthesis of high quality of such nanoparticle with < 100 nm hydrodynamic size is challenging and current approaches produce large size particles with significant loss of fluorescence property due to aggregation caused quenching issue. AIEgen molecules with aggregation-induced emission property offers new opportunity in development of such nanoparticle. Here we report AIEgenbased magnetic-fluorescent nanoparticle with varied surface chemistry and average hydrodynamic size between 25-50 nm. The approach involves covalent linking of soluble AIEgen to polymer coated colloidal magnetic nanoparticle surface that ‘switch on’ the AIEgen fluorescence. The resultant colloidal nanoparticle has cationic/anionic/zwitterionic surface charge with primary amine termination. Developed nanoparticle has been used as fluorescent cell imaging probe and magnetic separation of labelled cells. Presented approach can be extended for development of AIEgen-based various functional nanoparticle and nanobioconjugate.

KEYWORDS: nanoparticle, aggregation induced emission, bioimaging, magnetic separation, fluorescent probe


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INTRODUCTION Multifunctional nanoparticles have enormous application potential in biomedical and chemical science.1-21 Prominent applications include multimodal imaging,8,10,12 imaging and separation,17 cell tracking,12 drug and siRNA delivery21 and biosensing.9 A variety of multifunctional nanoparticles have been developed that include hybrid nanoparticle1-10 or fluorophore conjugated plasmonic/magnetic nanoparticle.11-13 These hybrid materials have magnetic-fluorescent,4,6,11,12,14-21

plasmonic-fluorescent1-3,7,9

and

magnetic-plasmonic5,10

properties. Among them nanoparticle with magnetic-fluorescent property is an interesting class that can be used for detection/imaging via fluorescence or magnetic resonance imaging (MRI) mode, as well as for magnetic separation application.14-21 Application of these nanoparticles towards biomedical application requires that they should be colloidal in nature with hydrodynamic size between 10-100 nm, appropriate surface chemistry and terminated with affinity biomolecules of interest.22 However, the synthesis of high quality magnetic-fluorescent nanoparticle with small hydrodynamic size and surface modification with different biomolecules is a challenging issue. In particular, hybrid nanoparticle based approach leads to larger hydrodynamic size and significant loss of fluorescence property.9,17 Similarly; fluorophore conjugation approach to magnetic nanoparticle leads to fluorescence quenching effect either due to aggregation between fluorophores23 or due to energy transfer from fluorophore to nanoparticle.13 Recently, a new class of molecules are reported with aggregation-induced emission (AIE) property commonly known as AIEgens.24-26 AIEgens are weakly emissive in molecularly soluble state as excited singlet state get relaxed through non-radiative low frequency vibration mode. However, under the solid/aggregated state the intramolecular rotations becomes restricted and exited state of AIEgen relaxes through radiative decay.24-26 This comparative advantage has been used to develop AIEgen-based multifunctional nanoparticle.27-31These 3 ACS Paragon Plus Environment


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multifunctional nanoparticles include AIEgen-plasmonic nanoparticle composite for multimodal imaging,27,28 AIEgen-magnetic nanoparticle composite for multimodal imaging,29 and AIEgen-graphene composite for imaging and phototherapy.30 However, hydrodynamic size of reported AIEgen-based multifunctional nanoparticles are > 100 nm in size with limited surface chemistry for functionalization and restricts wider biomedical application potential.2730

We design and develop nanoprobe for cellular/sub-cellular targeting, imaging and therapy.22,32 We use quantum dot,22 magnetic nanoparticle,22 plasmonic nanoparticle22 and AIEgens33,34 for development of these nanoprobes. We have designed nanoprobe for controlling endocytotic uptake mechanism and for targeting different subcellular compartments such as mitochondria, lipid droplet and perinuclear regions.22 We also develop multifunctional nanoprobe via hybrid nanoparticle approach9 and fluorophore conjugation approach to nanoparticle.13 Here we report AIEgen-based magnetic-fluorescent nanoparticle with 25-50 nm hydrodynamic size and variable surface chemistry. The approach involves ‘switching on’ of AIEgen fluorescence via its covalent linking to polymer coated colloidal magnetic nanoparticle surface. The resultant nanoparticle becomes magnetic-fluorescent with three distinct advantages over reported AIEgen-based multifunctional nanoparticle.27-31 First, the hydrodynamic size is small (average size < 50 nm) and suitable for cellular and subcellular targeting application. Second, nanoparticle surface charge can be varied to positive, negative or zwitterionic that can be used to control the cellular interaction. Third, nanoparticle surface is terminated with primary amines that can be used for conjugation chemistry and preparation of various functional nanoparticle and nanobioconjugates.


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EXPERIMENTAL SECTION Materials. Poly(ethylene glycol) methacrylate, 3-sulfopropyl methacrylate, N,N methylenebis-acrylamide, N-(3-aminopropyl)-methacrylamide hydrochloride, N, N methylene-bisacrylamide (MBA), ammonium persulfate,4-methyl morpholine-N-oxide (MNO), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

bis[2-

(methacryloyloxy)ethyl]phosphateand octadecene were purchased from Sigma-Aldrich and used as received. Details of synthesis of tetraphenylethene (TPE) is described elsewhere.33 Preparation of polyacrylate coated magnetic nanoparticle (MN control I/II/III). Hydrophobic γ-Fe2O3nanoparticle was synthesized via high temperature colloidal method.32 In brief, a mixture of 0.37 g iron stearate, 0.16 g octadecylamine, 0.16 g MNO and 8 mL octadecene was heated at 60 °C under inert atmosphere for 15 min. After that temperature of the reaction mixture was increased to 300 °C and continued heating at this temperature for 10 min. The nanoparticle was then purified from excess surfactants via acetone/ethanol-based precipitation

and

chloroform/cyclohexane-based

redispersion.

Hydrophobic

γ-Fe2O3

nanoparticle was then converted into hydrophilic nanoparticle by using polyacrylate coating as described earlier.13,32 Molar ratio of acrylate monomers were appropriately adjusted as described previously.13,32 In brief, 30 mg N-(3-aminopropyl)-methacrylamide hydrochloride was used for preparation of MN-control I; 14 mg N-(3-aminopropyl)-methacrylamide hydrochloride and 12 mg 3-sulfopropyl methacrylate were used for preparation of MN-control II; 9 mg N-(3-aminopropyl)-methacrylamide hydrochloride and 12 mg 3-sulfopropyl methacrylate were used for preparation of MN-control III. In all cases 6 μL bis[2(methacryloyloxy)ethyl]phosphate

was used as cross linker in addition to other acryl

monomers. Resultant polyacrylate coated γ-Fe2O3 nanoparticle was then washed with chloroform and ethanol repeatedly and dispersed in 3-5 mL distilled water. Finally,



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polyacrylate coated γ-Fe2O3nanoparticle solution was dialyzed using nitrocellulose membrane (MWCO12 000 Da) to remove unreacted reagents. Preparation of TPE conjugated magnetic nanoparticle (MN-TPE-I/II/III). 500 μL aqueous solution of MN-control I/II/III was diluted to one mL by adding methanol. In another vial, 2.0 mg TPE was dissolved in one mL methanol, mixed with nanoparticle solution and stirred for 15 min. After that, 100 μL aqueous solution of NaCNBH3 (10 mg/mL) was added and stirred for overnight. Next, resultant TPE conjugated nanoparticle (MN-TPE-I/II/III) was purified by dialysis followed by chloroform extraction to remove unbound TPE. In order to vary the number of TPE per nanoparticle, three different amounts of TPE (0. 5 mg, 1 mg, 2.0 mg) were dissolved in one mL methanol and used for conjugation with MN-control I by the processes described above. Colloidal solution of nanoparticle was used to measure optical property (using BioTek Synergy MX microplate reader) and hydrodynamic size and zeta potential (using Malvern, Nano ZS). A superconducting quantum interference device (SQUID) magnetometer was used for magnetic measurement study of the solid composite materials. Fourier transform infrared spectroscopy on KBr pellets was performed using Shimadzu FT-IR 8400S instrument. TEM study was performed using FEI Tecnai G2F20 microscope with a field-emission gun operating at 200 kV. FESEM was performed with a JEOL JSM-7610F microscope. Estimation of primary amine and TPE in nanoparticle. The number of primary amines per nanoparticle was determined before TPE conjugation via fluorescamine test. For this borate buffer solution (pH 9) of polymer coated nanoparticles and acetone solution of fluorescamine (one mg/mL) were prepared separately. Next, equal volume (100 μL) of fluorescamine solution and nanoparticle solution were mixed and then fluorescence was measured at an excitation wavelength of 400 nm. For determination of the number of TPE per particle, a standard calibration curve was prepared from the absorbance value of TPE molecule at 354 nm at


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different concentration. (Supporting Information, Figure S1) Next, the concentration of TPE present in the nanoparticle was determined by measuring the absorbance value at 354 nm and fitting it with the above mentioned calibration graph. Concentration of γ-Fe2O3 nanoparticle was determined by reported method.32 Cell labelling experiment. Human cervical cancer cell line (HeLa) was cultured as described earlier.34 For cell labelling study cells were seeded in 24 well plates with 500 µL culture media. After overnight growth, cells were treated with 100-200 µL of sample solution with final concentration 0.16 mg/mL. After one hour incubation washed cells were imaged using Olympus IX81 microscope attached with a digital camera. For colocalization study cells nanoparticle labelled cells were further incubated with fresh media for 24 h and then treated with lysotracker red for 30 min. Then washed cells were used for imaging study. For cytotoxicity study, cells were incubated with different doses of sample for 24 h. Then, cells were washed with PBS to remove unbound particles and fresh 500 μL culture media was added. Next, each well plate with attached cells was treated with 50 μL freshly prepared MTT solution (5 mg/mL) for 4 h. After that, supernatant was removed carefully leaving formazan in plate. Next, formazan was dissolved in freshly prepared sodium dodecyl sulphate (SDS) solution (8 g SDS dissolved in 40 mL 50 % water-DMF solution) and absorbance was measured at 570 nm. Cell viability was estimated assuming 100 % viability for control without any sample treatment.

RESULTS Conversion of magnetic nanoparticle to magnetic-fluorescent colloid via AIEgen conjugation. Synthetic approach of AIEgen conjugation to magnetic nanoparticle is shown in Scheme 1. First, surfactant capped hydrophobic γ-Fe2O3 nanoparticle of 6-10 nm size is synthesized via high temperature colloid-chemical approach and then it is converted into 7 ACS Paragon Plus Environment


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primary amine terminated hydrophilic nanoparticle via polyacrylate coating.13,32 The hydrophobic γ-Fe2O3 nanoparticle is insoluble in aqueous media due to presence of hydrophobic surfactants on their surface. So we have transformed them to polyacrylate coated nanoparticle where the hydrophilic shell provides colloidal stability. Next, primary amines are used for covalent conjugation with AIEgen molecule.33,34 We have used tetraphenylethene (TPE) molecule as AIEgen. The TPE molecule has one aldehyde group in its structure so that it can react with primary amine and form covalent bond. We have used three different types of polyacrylate shell and depending on the shell type, the TPE conjugated nanoparticles are named as MN-TPE-I, MN-TPE-II and MN-TPE-III. The MN-TPE-I is derived from MN-control I with the shell having primary amine (NH2) and polyethylene glycol. Similarly, MN-TPE-II and MN-TPE-III are derived from MN-control II and MN-control III, respectively, both of which has NH2, PEG and sulfopropyl (SO3-). The only difference between MN-control II and MNcontrol III (and hence MN-TPE-II/MN-TPE-III) is the different % of functional groups.Presence of primary amines on the surface of MN-control I/II/III has been estimated via fluorescamine test. The tentative numbers of primary amines per particles are 600, 450 and 300 for MN-control I, MN-control II and MN-control III, respectively. In addition the tentative number of TPE present per MN-TPE-I/II/III has been determined from the absorbance of TPE at 354 nm. The values are 230, 230 and 120 for MN-TPE-I, MN-TPE-II and MN-TPE-III, respectively. These values indicate a fraction of primary amines are reacted and the remaining numbers of primary amines are 370, 220 and 180 for MN-TPE-I, MN-TPE-II and MN-TPEIII, respectively. Physical properties of all nanoparticles are summarized in Table 1, Figure 1-4 and Supporting Information, Figure S2, S6, S7. Figure 1a shows the AIE property of TPE. As the water is added to the ethanol solution of TPE, molecularly soluble TPE starts aggregating that result the ‘switch on’ of their green fluorescence. Similar type fluorescence ‘switch on’


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property is also observed after conjugation of TPE with polymer coated magnetic nanoparticle. (Figure 1b and Supporting Information, Figure S2) In particular it is shown that as the number of attached TPE per particle increases from 60 to 230, the ‘switching on’ property of TPE is enhanced. In contrast molecularly soluble TPE solution of similar concentration is completely non-fluorescent. (see Figure 1a) This result clearly suggests that a dominant TPE-TPE interaction occurs at the nanoparticle surface that leads to their fluorescence switch on property. As the fluorescence ‘switching on’ property of TPE increases with increased number per nanoparticle, we have tried to conjugate maximum number of TPE per particle with the maximum TPE-TPE interaction. Figure 2 shows UV-visible absorption spectra of MN-TPE-I, MN-TPE-II and MNTPE-III with the characteristic absorption band of TPE at 325 nm and emission band between 450-550 nm. However, the absorption band and emission band maxima of TPE in MN-TPEI/II/III blue shifts from 354 nm to 325 nm and 530 nm to 500 nm, respectively, as compared to ethanol-based TPE aggregates. This is due to the transformation of –HC=O group of TPE to – H2C NH– group that minimize π-conjugation. The fluorescence quantum yield has been measured using quinine sulphate as reference and the values are in the range of 6-8 % for MNTPE-I/MN-TPE-II and 3-4% for MN-TPE-III. (Table 1) The lower quantum yield value for MN-TPE-III is due to lowers number of TPE per particle with lower TPE-TPE interaction. Polyacrylate coating on the nanoparticle surface has been verified by FourierTransform Infrared (FTIR) spectra. FTIR spectroscopy of MN-control I and MN-TPE-I shows appearance of hydroxyl group signals around 3000-3500 cm-1 due to polyacrylate component. (Supporting Information, Figure S3). TEM image shows inorganic γ-Fe2O3 core of 6-10 nm and polymer shell is observed as light grey color around dark core after the uranyl acetate staining. (Supporting Information, Figure S4) SEM image show overall size of 15-22 nm that include polyacrylate coating and core nanoparticle. (Supporting Information, Figure S5)



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Hydrodynamic sizes of nanoparticles is measured before and after TPE conjugation. Result shows that all nanoparticles are 25-50nm in average hydrodynamic size, including inorganic core and polymer shell along with all chemical functional groups. (Figure 2) Surface charge of the nanoparticles is determined at different solution pH by zeta potential measurement. (Table 1) It is observed that MN-TPE-I shows positive surface charge at pH 4.5 due to protonated NH2(NH3+) and as the pH increases to 7-9, the charge becomes near zero value due to deprotonation of NH2 groups. We called this nanoparticle as cationic as it shows positive charge in the pH range of 4.5-7.4. In contrast MN-TPE-II shows relatively lower positive surface charge at pH 4.5 as NH3+ ions are partially balanced by anionic sulfopropyl (SO3-) groups and as the pH increases to 7.4, the charge approaches to near zero value but at pH 9.0 the surface charge becomes anionic. We called this nanoparticle as zwitterionic as it changes the surface charge from positive to negative, as the pH changes from 4.5 to 9.0. In contrast surface charge of MN-TPE-III shows negative value in all pH, due to dominant SO3- groups and we termed it as anionic nanoparticle. Magnetic property measurement system (MPMS) has been used to study the magnetic property of MN-TPE-I and MN-control I. (Figure 3) Result shows characteristic hysteresis, similar to superparamagnetic materials, which almost disappears at room temperature. The saturation magnetization values for MN-control I are 3.8 and 2.9 emug-1 at 10 K and 300 K, respectively. Similarly, for MN-TPE-I the saturation magnetization values are 3.4 and 2.5 emug-1 at 10 K and 300 K, respectively. The zero field cooling (ZFC) and field cooling (FC) magnetization curve of MN-control I and MN-TPE-I shows a well-defined blocking temperature at 20 K and 10 K respectively, which also indicate superparamagnetic nature of the nanoparticle. Colloidal stability of MN-TPE-IMN-TPE-II and MN-TPE-III has been investigated in buffer solution of varied pH. Digital images of all colloidal dispersions are shown in Figure 4.


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The result shows that nanoparticles have good colloidal stability in all buffer solution for more than one month. However, colloidal nanoparticles can be precipitated by adding large amount of salt and precipitated particles can then be attracted by laboratory-based bar magnets. (Figure 4) Magnetically separated particles can be redispersed in fresh aqueous media or by removing of salt via dialysis. Fluorescence stability study of MN-TPE-I, MN-TPE-II and MN-TPE-III has been carried out in presence of buffer solution of different pH. (Supporting Information, Figure S6) Results show that fluorescence intensity of MN-TPE-I remains intact at pH 7.4 and 9.0 but decreases to some extent at pH 4.5. This may be due to protonation of primary/secondary amine group that reduces restricted intramolecular motion (RIM) to a certain extent. Fluorescence stability of MN-TPE-I, MN-TPE-II and MN-TPE-III is further investigated under continuous UV exposure of colloidal solution of nanoparticles and drop casted film of nanoparticle which show that fluorescence remain intact under continuous light exposure of 30 min. (Supporting Information, Figure S7) These results suggest that fluorescence of nanoparticles would be stable under continuous excitation and useful for long term imaging application.

AIEgen conjugated magnetic nanoparticle as magnetic-fluorescent cell imaging probe. In order to explore the bioimaging application potential, MN-TPE-I, MN-TPE-II and MN-TPEIII have been used to label cells. Typically, HeLa cells are incubated with nanoparticle for one hour and washed cells are incubated with fresh media for 24 h. Next, washed cells are imaged under fluorescence mode with UV excitation. Results are summarized in Figure 5 and Supporting Information, Figure S8. It is observed that MN-TPE-I and MN-TPE-II can label cells within one hour and their fluorescence intensity within cell remain intact even upto 24 h and they localize in cell cytoplasm. (Figure 5) In contrast labeling efficiency of MN-TPE-IIIis



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relatively poor and their fluorescence intensity inside cells decreases with longer incubation time. It is observed that localization of MN-TPE-I/MN-TPE-II inside cell takes 8-24 h depending on the nature of surface charge. In order to investigate multifunctional application potential of these nanoparticle, MN-TPE-I labelled cells are dispersed in cell culture media and kept under laboratory-based bar magnet. It can be found that labelled cells are easily separated by magnet. (Figure 5a) In order to understand the nature of localization, co-localization study with lysotracker red has been performed. It has been found that particles significantly colocalize with lysotracker red. (Supporting Information, Figure S8) This result suggests that nanoparticles enter into cell via endocytosis processes that traffics them to endosome/lysozome and different extent of labeling is linked to the surface property of nanoparticle. MN-TPE-I with cationic surface charge has strong interaction with anionic cell membrane but MN-TPEII with zwitterionic surface charge has modular interaction to cell membrane and MN-TPE-III with anionic charge can only weakly interact to membrane via lipophilic TPE groups. Thus, MN-TPE-III has weaker labelling property than other two nanoparticles. Cell viability study of MN-TPE-I/II/III nanoparticles has been performed using HeLa cells via MTT assay. Result shows low cytotoxicity of all three nanoparticles at wider concentration range. (Supporting Information, Figure S9) Although cationic nanoparticles are known to have good labelling property along with cytotoxic effect,22 we have intentionally designed MN-TPE-I with lower cationic charge and PEGylated functional groups. This allows MN-TPE-I with high cell uptake with lower cytotoxicity.

DISCUSSION AIE active hydrophobic TPE molecule is non-emissive in organic solvents such as methanol/ethanol in which it remain in molecularly soluble form. In aqueous environment it 12 ACS Paragon Plus Environment

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started to aggregate where intramolecular rotation of phenyl rotors becomes restricted and ‘switches on’ its fluorescence with increasing water volume % in water-ethanol mixture. Here we have used the strategy of covalent conjugation of TPE to polymer coated magnetic nanoparticle surface to ‘switch on’ its fluorescence. Transformation of non-fluorescent magnetic nanoparticle into fluorescent one by this type covalent conjugation with conventional organic fluorophore has significant limitations due to the fluorescence quenching issue via intermolecular energy transfer. In contrast AIE active TPE molecule does not have such quenching issue.13,23 Presented approach involves covalent conjugation of TPE to the primary amine terminated magnetic nanoparticle. Considering the fact that nanoparticles are 25-50 nm in hydrodynamic size and each nanoparticle has 120-230 TPE, it can be expected that TPEs are close to each other at the nanoparticle surface that offers TPE-TPE interaction that leads to restriction in intramolecular motion (RIM) and fluorescence ‘switch on’ phenomenon. However, this TPE-TPE interaction is too weak to offer any red/blue shifting of their UV visible absorption spectrum. Presented synthetic approach has three additional advantages coming from polyacrylate coating. First, nanoparticle can be prepared with good colloidal stability as compared to other coating approaches. For example we have demonstrated good colloidal and fluorescence stability of three designed nanoparticles under physiological conditions. Second, nanoparticle with wide variation of surface chemistry can be generated using different acryl monomers and by varying their molar ratio. For example, we have prepared three different nanoparticle with cationic, anionic and zwitterionic surface charge. Third, nanoparticle can be further functionalized using the remaining primary amines on their surface. In particular poor cell labelling property of MN-TPE-III is ideal to minimize non-specific labelling of nanoparticle and can be used for selective labelling after conjugation with bioaffinity molecules.



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CONCLUSION We have shown that magnetic-fluorescent colloidal nanoparticle with average hydrodynamic size of 25-50 nm can be prepared via covalent linking of AIEgen to polymer coated magnetic nanoparticle surface. This processes involves the ‘switching on’ of AIEgen fluorescence which is otherwise non-fluorescent in molecularly soluble state. The resultant colloidal nanoparticle has cationic/anionic/zwitterionic surface charge with primary amine termination. It is demonstrated that the nanoparticle can be used as cell imaging probe and labelled cells can be magnetically separated. The primary amines present at the nanoparticle surface can be used for the development of various functional nanoparticle and nanobioconjugates.

ASSOCIATED CONTENT Supporting Information Details of quantification of primary amines in the nanoparticle surface, TEM image of polymer coated magnetic nanoparticle, colocalization study of nanoparticle with lysotracker and cytotoxicity data. This material is available free of charge via the Internet at http://pubs.acs.org. Notes The authors declare no competing financial interests. Acknowledgement. NRJ acknowledge DST Nano Mission (Grant number SR/NM/NB/ 1009/2016) and CSIR (Grant number 02(0249)15/EMR-II) Government of India for financial assistance. K.M. acknowledges CSIR, India for providing research fellowship.


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Table 1. Property of AIEgen-derived magnetic-fluorescent nanoparticles. Nanoparticle type

Functional groups (number per particle)#

MN-TPE-I MN-control I MN-TPE-II MN-control II MN-TPE-III MN-control III

-NH2(370), TPE (230) -NH2(600) -NH2(220), -SO3-, TPE(230) -NH2(450),-SO3-NH2(180),-SO3-, TPE(120) -NH2(300), -SO3-

#Each

Average hydrodynamic size, fluorescence quantum yield (%) 25 ± 10 nm, 6-8 % 25 ± 10 nm, -----35 ± 10 nm, 6-8 % 25 ± 10 nm, -----50 ± 20 nm, 3-4 % 50 ± 20 nm, ------

Surface charge at pH 4.5 7.4 9

Cell labelling performance, cytotoxicity

+23 +4 -6 +18 +4 -4 +9 -1 -13 +8 0 0 -2 -13 -15 -1 -3 -10

good, low good, high good,low good, low poor, low poor, low

nanoparticle has PEG functional groups in addition to mentioned functional groups.



Page 20 of 26

Scheme 1. Synthetic strategy for AIEgen conjugated magnetic-fluorescent nanoparticle.It involves transformation of 6-10 nm hydrophobic γ-Fe2O3nanoparticle to primary amine terminated hydrophilic nanoparticle of 25-50 nm hydrodynamic size via polyacrylate coating followed by conjugation with AIEgen (TPE). Depending on the acrylate monomer used nanoparticles are cationic (MN-control I/MN-TPE-I), zwitterionic (MN-control II/MN-TPEII) and anionic (MN-control III/MN-TPE-III) in nature.

++++ +++ ++ + + ++ + + + + + + + ++ + ++ + ++++++++

+

-++-++- ++ -++ + + - ++-++-++-

NaCNBH3

+ +

+

+

+

+

+

-

+

/ (off) (on) H3CO

TPE

OCH3

MN-TPE-I

NaCNBH3

+

+

magnetic core

+

-

+

-

polyacrylate shell

MN-TPE-II

MN-control II

--++- ++ --+ + + + -++ -- + +-+++ --+ MN-control III

CHO

+

MN-control I

++ ++ + + +

+ +

++

NaCNBH3

+ -- + --+ -+ -MN-TPE-III

-

NH2/NH3+ SO3PEG


1.5

Absorbance

80

500

60

0.9 0.6

50

400

40

300

20

200

0

0.3

b)

600

100

1.2

0.0

700

water (%)

100 300

400

500

600

0 700

Wavelength (nm) 1.0

1000 3 2 1

0.8

800

0.6

600

0.4

400

0.2

200

0.0

300

400

500

600

0 700

Fluorescence Intensity (a.u.)

a)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 21 of 26

Wavelength (nm)

Figure 1. a) Aggregation-induced emission property of TPE as observed by precipitation of same concentration of TPE with increasing water volume % in the water-ethanol mixture. b) Evidence of ‘switching on’ of TPE fluorescence after binding with colloidal magnetic nanoparticle. Varied amount of TPE is conjugated with MN-TPE-I and average number of TPE per particle is 60 (1), 120 (2) and 230 (3).TPE concentration is kept same (by adjusting same absorbance of TPE at 325 nm for three samples), in order to show that ‘switching on’ property appears with increased number of TPE per particle.


1.0 800

Absorbance

0.8 600

0.6

400

0.4

200

0.2 0.0

300

400

500

600

0 700

MN-control I

25

MN-TPE- I

20 15 10 5 0

0

600

0.6 0.4

200 0.2 0.0

300

400

500

600

0 700

10 5 0

1000

30

0.8

800

25

0.6

600

0.4

400

0.2

200 400

500

600

0 700

MN-TPE- II

15

1.0

300

150

MN-control II

20

Wavelength (nm)

0.0

100

Size (nm)

25

Number (%)

c)

400


Absorbance

0.8

50

30

Number (%)

1.0


Wavelength (nm)

b)

Page 22 of 26

30

Number (%)

a)

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



0

50

100

Size (nm)

150

MN-control III MN-TPE- III

20 15 10 5 0

0

Wavelength (nm)

50

100

Size (nm)

150

Figure 2. a) Absorption/emission spectra and dynamic light scattering-based hydrodynamic size MN-TPE-I (a), MN-TPE-II (b) and MN-TPE-III (c).


a)

b) Magnetization (emu/g)

i)

4 3

10 K 300 K

2 1 0 -1 -2 -3 -4

-40000 -20000

0

1.5 1.0 0.5 0

50

100

150

200

250

300

3

10 K 300 K

2 1 0 -1 -2 -3

-40000 -20000

0

20000 40000

Field (Oe) 2.5

Magnetization (emu/g)

FC ZFC

2.0

4

-4

ii)

2.5

0.0

i)

20000 40000

Field (Oe)

ii) Magnetization (emu/g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Magnetization (emu/g)

Page 23 of 26

FC ZFC

2.0 1.5 1.0 0.5 0.0

0

Temperature (K)

50

100

150

200

250

300

Temperature (K)

Figure 3. Field dependent magnetization curves of MN-control I (a) and MN-TPE-I (b) at 10 K and 300 K and the respective temperature dependent zero-field-cooled (ZFC) and fieldcooled (FC) magnetization curves.



magnet

Magnetic separation

magnet

pH 9

MN-TPE-I

pH 7.4

MN-TPE-II

pH 4.5

magnet

MN-TPE-III

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

Figure 4. Colloidal solution of MN-TPE-I,MN-TPE-II and MN-TPE-III in daylight or under handheld UV light (UV) in different buffer solutions. Right panel shows the magnetic separation of nanoparticles by using laboratory-based bar magnet after their precipitation.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a) 1h

magnet

Page 25 of 26

24 h

50 microns

50 microns

b) 1h

24 h

50 microns

c)

50 microns

24 h

1h

50 microns

50 microns

Figure 5. Application of MN-TPE-I (a), MN-TPE-II (b) and MN-TPE-III (c) as fluorescent cell imaging probe. Inset shows magnetic separation of MN-TPE-I labelled cells. HeLa cells are incubated with sample for 1 h and then washed cells are incubated with fresh culture media upto 24 h and imaged under bright field or fluorescence mode and merged images are shown here. Results show that of MN-TPE-I and MN-TPE-II can label cells very efficiently as compared to MN-TPE-III and stay/localize inside cytoplasm in next 24 h.For magnetic separation, cells are detached from culture plate (via tripsin-EDTA) and then water dispersed cells are observed under hand held UV light in absence/presence of magnet. 25 ACS Paragon Plus Environment


TOC

50 micron cell imaging

H3CO

magnetic core,

OCH3

polymer shell,

magnet

CHO

magnet

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 26

polyethylene glycol


AIEgen-Conjugated Magnetic Nanoparticles as MagneticâFluorescent

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