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Galactose Functionalized, Colloidal-Fluorescent Nanoparticle from Aggregation Induced Emission Active Molecule via Polydopamine Coating for Cancer Cell Targeting Kuheli Mandal, and Nikhil R. Jana ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00673 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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ACS Applied Nano Materials

Galactose Functionalized, Colloidal-Fluorescent Nanoparticle from Aggregation Induced Emission Active Molecule via Polydopamine Coating for Cancer Cell Targeting Kuheli Mandal and Nikhil R. Jana* Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata 700 032, India *Address for correspondence to [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT: Aggregation induced emission (AIE) active molecules emerge as new generation fluorescent probes and their transformation to functional nanoparticle can greatly extend the application potential. Here we report an approach for transforming AIE active tetraphenylethene (TPE) molecule to galactose functionalized colloidal nanoparticle that can be used for targeting/labelling of cancer cells. The approach involves polydopamine coating around aggregated TPE or TPE-chitosan conjugate followed by covalent linking with thiolated galactose. Resultant nanoparticles have 50-200 nm hydrodynamic size with good colloidal stability at physiological condition, 6-12 % fluorescence quantum yield and they selectively label cells with over expressed galactose receptors. The unique advantage of this nanoprobe is that fluorescence remains intact in presence of conventional quenchers. This approach may be extended for making AIE-active molecule-based various functional nanoprobes. KEYWORDS: nanoparticle, aggregation induced emission, polydopamine coating, imaging probe, bioconjugation


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INTRODUCTION Fluorescent nanoparticles are widely used as bioimaging probes for monitoring biochemical activities at cellular, subcellular and single molecule length scale.1-5 Fluorescent nanoparticles are designed from variety of inorganic materials such as quantum dot,1-3 fluorescent silicon nanoparticle,6 fluorescent carbon nanoparticles,7 doped semiconductor nanocrystals8 and fluorescent metal nanoclusture.9 Although great advances have been made in designing such nanoprobes, their in vivo application is limited due to non-biodegradability and cytotoxicity.10 In contrast fluorescent organic molecule-based probes11-13 are smaller in size for easy clearance from living body as compared to nanoparticle.14 However, organic fluorophores need to be transformed to nanoparticle structures using polymer/surfactant components and such transformation is limited because of fluorescence quenching effect of molecular fluorophores during aggregation.13 Weakly emitting/non-emitting molecules with aggregation induced emission (AIE) property can solve this quenching issue in designing organic fluorophore-based nanopartcile.15-18 The AIE property of molecules appears due to restriction of intramolecular rotation/vibration at aggregated states and under molecularly soluble state such rotation/vibration is allowed that makes AIE molecules nonemissive.15,16 Varieties of AIE molecules are synthesized and they have

been

used

as

fluorescent

‘switch

on’

probe

for

detection/imaging

of

biomolecule/biochemical activities.15,16 In addition they have been transformed to fluorescent nanoprobe for extended biomedical application.17,18 There are two common approaches in making AIE molecule based nanoparticle. In first approach AIE molecule is incorporated into aggregated polymer nanoparticle/micelle19-21 and in second approach AIE molecule-based self-assembled molecule/polymer is designed.22,23 We have recently reported a new approach in making AIE molecule based nanoparticle that involves aggregation of AIE molecule in presence of functional AIE molecule.24 Although this approach is more powerful than other 3 ACS Paragon Plus Environment


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approach in making < 100 nm functional nanobioconjugate, it has two common limitations. First, the fluorescence signal of self-assembled structure is unpredictable under complex biological condition due to unpredictable nature of assembly. Second, this approach requires chemical

synthesis

of

specialized

molecule

with

the

finer

adjustment

of

hydrophobic/hydrophilic balance for self-assembly processes. Thus new approach in making AIE molecule based robust functional nanoparticles with 10-200 nm hydrodynamic size can greatly facilitate their application potential. Here we report colloidal functional nanoparticle from tetraphenylethene (TPE)-based AIE molecule via polydopamine coating approach. Dopamine undergoes oxidative selfpolymerization under mild alkaline condition and resultant polydopamine can spontaneously deposit on various surface.25-28 Polydopamine has been used as biocompatible and biodegradable coating for organic/inorganic material29,30 and nanoparticle/microparticle31-35 but not utilized in making AIE molecule based nanoparticle. The polydopamine coating thickness can be controlled by varying the concentration of dopamine and reaction time and redox property of catechol groups in polydopamine can be used for functionalization with amine/thiol terminated biomolecule through Schiff base or Michael addition reaction.36,37 In the presented approach aggregated TPE or TPE-chitosan is coated with polydopamine to prepare colloidal nanoparticle and then conjugated with thiolated galactose via Michael addition reaction. Following this approach galactose functionalized fluorescent nanoprobe of 50-200 nm size have been synthesized and used for selective labelling of cells with over expressed galactose receptors.

EXPERIMENTAL SECTION Materials.

Dopamine

oligosaccharide

lactate,

hydrochloride,

lactose,

cysteamine

hydrochloride,

O-[2-(6-oxocaproylamino)ethyl]-O'-methylpolyethylene

chitosan glycol


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(PEG-CHO) (Mw 2000) are purchased from Sigma-Aldrich and used as received. Details of synthesis of tetraphenylethene (TPE) is described elsewhere.38 Synthesis of polydopamine coated TPE nanoparticle. About 0.5 mg TPE was dissolved in 2 mL chloroform solution. In a separate vial ethanol-water solution was prepared by mixing 2 mL aqueous phosphate (10 mM) buffer solution of pH 8.5 with 2 mL ethanol. The ethanolwater solution was kept under vigorous stirring condition and then whole TPE solution was added. The stirring was continued for 4-5 h until all chloroform was evaporated with the formation of colloidal AIE nanoparticles. Next, 5 mg dopamine was dissolved in 100 µL water and added to colloidal AIE nanoparticles. The solution mixture was stirred for 12 h and polydopamine coating was continued for 12 h. Next, the solution was centrifuged at 6,000 rpm for 5 min to remove larger aggregates and further purified by dialysis for 24 h using nitrocellulose membranes (MWCO 12 kDa) to remove ethanol. Finally nanoparticles are precipitated by centrifugation at 12,000 rpm and redispersed in 2.0 mL water with the TPE concentration range of 0.5 mg/mL. Synthesis of polydopamine coated TPE-chitosan nanoparticle. About 2 mg chitosan oligosaccharide lactate was dissolved in 0.5 mL distilled water and mixed with 0.5 mL aqueous solution of PEG-CHO (0.8 mM). The mixed solution was stirred for 1 h and then the solution was diluted by adding 1.0 mL methanol. Next, 1 mL methanolic solution of TPE (2 mg/mL) was added and stirred for 15 min. Then, 100 µL aqueous solution of NaCNBH3 (4 mg/mL) was added. After overnight stirring the total solution was mixed with 2 mL aqueous phosphate (10 mM) buffer solution of pH 8.5, kept under overnight stirring for evaporation of most of the methanol. Next, polydopamine coating was initiated by adding 100 µL of aqueous solution of dopamine (1.0 mg/mL) and stirring was continued for 2-3 h. Next, the solution was centrifuged at 10,000 rpm for 5 min and precipitated larger aggregates were removed. Supernatant containing nanoparticle was dialyzed for 24 h using nitrocellulose



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membranes (MWCO 12 kDa) in order to remove methanol. Finally nanoparticles are separated by centrifugation at 14,000 rpm and redispersed in 2.0 mL water with the TPE concentration range of 1 mg/mL. Synthesis of thiolated galactose. About 9 mg lactose was dissolved in 0.5 mL borate buffer solution of pH 9.0, mixed with 2 mg cysteamine (dissolved in 0.5 mL borate buffer of pH 9) and stirred for 15 min. Next, 50 mg solid NaCNBH3 was added and after overnight stirring thiolated galactose was precipitated by adding excess acetone followed by repeated washing. Finally precipitate was dissolved in 1.0 mL water. Galactose functionalization of polydopamine coated nanoparticle. Freshly prepared polydopamine coated TPE nanoparticle or TPE-chitosan nanoparticle was dispersed in PBS buffer solution of pH 8.5 and then 100 µL thiolated galactose solution was added. The stirring was continued for 3 h and functionalized nanoparticles were purified by centrifugation at 12000 rpm followed by washing three times with water and finally dispersed in 1.0 mL water. Presence of galactose in the functional nanoparticle was confirmed via anthrone test. Typically, anthrone solution (2 mg/mL) was prepared in 80 % conc. H2SO4. Then, 100 µL colloidal solution of as prepared nanoparticle was mixed with 2 mL anthrone solution and heated in water bath for 15 min at 90 °C. Next, the mixture was cooled in ice water and absorption spectra were measured. Fluorescence quenching study. Solutions of Cu2+, Fe3+, Fe2+, Zn2+ were prepared separately by dissolving the respective salts in distilled water. Buffer solution of pH 4.5 was prepared using acetate buffer, pH 7.4 was prepared using phosphate buffer and pH 9.0 was prepared using bicarbonate buffer. Water soluble silica coated Ag and Au nanoparticles were synthesized using reported method.39 Hydrophobic quantum dot (ZnS capped CdSe) was


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synthesized via high temperature colloid-chemical method and then coated with polyacrylate using our reported approach.40 The fluorescence quenching experiment was performed using 200 µL phosphate buffer solution (pH 7.4 of quantum dot or AIE-based nanoparticle (0.05-0.1 mg/mL) and separately mixing with 5-20 µL of metal nanoparticle or metal salt solution. Final concentrations of Cu2+, Fe3+, Fe2+, Zn2+ were maintained at 0.1 mM and Ag/Au concentration in their nanoparticle was maintained at 0.5 mM. Fluorescence quenching study in buffer solution of pH 4.5/9.0 was performed by preparing the nanoparticle solution in the respective buffer solutions. Fluorescence spectra were measured by exciting at 430 nm and 370-390 nm for quantum dot and AIE-based nanoparticle, respectively. Cell labelling experiment. Human liver cancer cell line (HepG2) was cultured in Dulbecco’s Modified Eagle’s medium (DMEM) media with 10 % fetal bovine serum and 1 % penicillin streptomycin at 37 °C and 5 % CO2 atmosphere. For cell labelling study cells were seeded in 24 well plates with 500 µL DMEM media. After overnight growth, cells were treated with 50 µL of colloidal nanoparticle solution with final concentration of 0.01 mg/mL. After 1 h incubation cells were washed with PBS buffer for two times to remove unbound particles. Finally, 500 µL fresh DMEM media was added and used for microscopic studies. Procedure for cytotoxicity study. For cell viability study, HepG2 cells were cultured in a 24 well plate in DMEM media. After that, cells were treated with different doses of samples for 24 h and then washed thoroughly with PBS buffer. After adding fresh DMEM media, each well plate with attached cells was treated with 50 µL of freshly prepared methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution (5 mg/mL) and incubated for 4 h. Then the supernatant was removed carefully leaving the formazon in the plate. This formazon was dissolved in freshly prepared SDS solution (8 g of SDS dissolved in 40 mL of 50 % DMF−H2O mixture), and absorbance of the solution was measured at 570 nm. Cell



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viability was estimated assuming 100 % viability for control sample without any AIE nanoparticle. Instrumentations. UV-visible absorption spectra were measured on a Shimadzu UV-2550 spectrophotometer using a quartz cell of 1 cm path length and fluorescence spectra were recorded on PerkinElmer fluorescence spectrometer model LS-45. Hydrodynamic size and zeta potential of nanoparticle were measured using Malvern (Nano ZS) instrument. Fluorescence images of cells were captured using Olympus IX81 microscope attached with a digital camera. High resolution mass spectra (HRMS) were recorded on a Waters QTOF Micro YA263 spectrometer. FESEM was performed with a Supra 40 (Carl Zeiss) microscope. Fourier Transform Infrared Spectroscopy (FTIR) on KBr pellets was performed using Shimadzu FT-IR 8400S instrument.

RESULTS Synthesis of TPE-based nanoparticle and galactose functionalization. The synthesis strategies of nanoparticles are shown in Scheme 1, 2 and their physical properties are summarized in Table 1. We have used two approaches for making AIE-based nanoparticle. We have used TPE molecule which has one aldehyde group in its structure. In approach I, chloroform solution of tetraphenylethene (TPE) was injected into 50 % water-ethanol mixture of pH 8.5 under stirring condition. Initially, the chloroform remains immiscible and mixture remains non-fluorescent. After the evaporation of chloroform, the mixture turns into optically transparent solution with green fluorescence, suggesting the formation of aggregated TPEbased particles. Polydopamine coating is then initiated on the particle surface by adding dopamine solution. The solution colour turns black within few hours, indicating the polydopamine formation. Next, polydopamine coated nanoparticles (TPE@PD) are isolated


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by centrifuge, separated from organic solvents by dialysis and finally dispersed in water. This approach can produce TPE@PD of 100-200 nm size. In approach II, TPE is conjugated with chitosan and then used for making nanoparticle. Specifically chitosan oligosaccharide, TPE and PEG-CHO are mixed in 1:10:2 molar ratio in water-methanol (1:2 volume ratio) mixture. Under this condition, primary amine groups of chitosan oligosaccharide react with aldehyde groups of TPE and PEG-CHO. The weekly emissive solution becomes green fluorescent after partial evaporation of methanol, indicating the formation of TPE-based aggregated colloidal solution. Next, polydopamine coating was initiated by adding dopamine solution. The solution becomes black after 2-3 h, indicating polydopamine coating. Resultant polydopamine coated nanoparticles (TPE-Chi@PD) are isolated by centrifuge and dialyzed to remove organic solvents and finally dispersed in water. We have tried various control experiments in these two approaches in order to decrease the particle size. (see Supporting Information, Table S1, S2 and Figure S1, S2) In approach I, five control experiments are performed. In control 1, ethanol solution of TPE is injected into 100 % water that produces micron size fluorescent aggregates. In control 2, ethanol solution of TPE is injected into water-ethanol mixture of 3:2 volume ratio and produced particle precipitate after partial evaporation of ethanol. In control 3, chloroform solution of TPE is injected into 100 % ethanol but no particle formation is observed and the solution remains non-fluorescent even after partial evaporation of solvent. In control 4, chloroform solution of TPE is injected into water-ethanol mixture of 3:2 volume ratio. Initially the solution remains non-fluorescent and becomes fluorescent after some time with partial precipitation of particles but polydopamine coated particles become colloidally unstable. In control 5, chloroform solution of TPE is injected into aqueous Tween 20 solution that produces fluorescent colloidal particle of < 50 nm size but polydopamine coating becomes unsuccessful on those particles surface. Similarly, two control experiments are performed in



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approach II. In control 6, chitosan oligosaccharide, TPE and PEG-CHO are used in 1:10:0 molar ratio in water-methanol mixture that results fluorescent precipitates of particles. In control 7, chitosan oligosaccharide, TPE and PEG-CHO are used in 1:28:2 molar ratio in water-methanol mixture but fluorescent precipitates are observed after partial evaporation of methanol. Galactose functionalization of TPE@PD and TPE-Chi@PD is performed using thiolated galactose. We have used lactose, a disaccharide of galactose and glucose, for galactose functionalization. At first, lactose is transformed to thiolated lactose by conjugating terminal aldehyde group of glucose with amine group of cysteamine. (Scheme 2a, Supporting Information, Figure S3). Next, thiol groups are used for Michael addition reaction with alkene groups present in polydopamine shell of TPE@PD and TPE-Chi@PD. Galactose functionalized resultant nanoparticles (TPE@PD-gal and TPE-Chi@PD-gal) are then preserved at 4 °C as stock solution. Galactose functionalization of nanoparticle has been confirmed by anthrone test that shows absorption band at 620 nm. (Supporting Information, Figure S4). Colloidal, fluorescent nanoparticle from TPE via polydopamine coating. Properties of TPE@PD, TPE-Chi@PD, TPE@PD-gal and TPE-Chi@PD-gal are summarized in Table 1 and Figures 1, 2, 3. TPE and TPE-Chi are weakly fluorescent under molecularly soluble state but shows AIE property under aggregated states. For example, they are non-fluorescent in methanol due to high solubility but show green fluorescence with increasing water volume % of the medium due to insoluble aggregate formation. (Figure 1) Similarly, TPE/TPE-Chibased nanoparticles exhibit green fluorescence due to the presence of aggregated TPE inside nanoparticle. Optical properties of nanoparticles are shown in Figure 2. The UV–visible absorption spectra of colloidal nanoparticles show light yellow colour with distinct band at 400 nm and after


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polydopamine coating the colour becomes brown with the increased absorbance in the 450700 nm regions. All the nanoparticle dispersions show green fluorescence. The nanoparticle made of TPE@PD and TPE@PD-gal show strong fluorescence band between 450-650 nm with the fluorescence maxima at 530 nm, under the 390 nm excitation. Similarly, nanoparticle made of TPE-Chi@PD and TPE-Chi@PD-gal show strong fluorescence band between 400-600 nm with the fluorescence maxima at 500 nm, under the 370 nm excitation. Control experiments show insignificant fluorescence by polydopamine based colloidal particles. (Supporting Information, Figure S5) Fluorescence quantum yield has been measured using quinine sulphate as reference and the values are in the range of 10–12 % for TPE@PD and TPE@PD-gal and 6-8 % for TPE-Chi@PD and TPE-Chi@PD-gal. (Table 1) Polydopamine coating on the nanoparticle surface has been verified by Fourier-Transform Infrared (FTIR) spectra, increased particle size from dynamic light scattering (DLS) and SEM study. FTIR spectroscopy of TPE@PD shows characteristic C-H vibration of aldehyde groups at 2840 cm-1 and 2900 cm-1due to TPE component and secondary amine and hydroxyl group signals around 3000-3700 cm-1 due to polydopamine component. (Supporting Information, Figure S6). SEM and DLS study show that size of TPE@PD increases from 6070 nm to 100-200 nm after the polydopamine coating and size of TPE-Chi@PD increases from 20-30 nm to 50-100 nm after the polydopamine coating. (Figure 3, 4 and Supporting Information, Figure S7.) In addition no significant change of particle size is observed after galactose functionalization. Surface charge of nanoparticles has been determined using zeta potential measurement at different solution pH. Results show that TPE@PD and TPE@PD-gal show low negative charge that changes from near zero value to –16 mV as the pH increases from 4.5 to 9.0. Similarly, zeta potential of TPE-Chi@PD and TPE-Chi@PD-gal changes from low positive to low negative value as pH increases from 4.5 to 9.0. Low anionic surface charge is due to



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the non-ionic functional groups present at the polydopamine surface. Cationic surface charge for TPE-Chi@PD and TPE-Chi@PD-gal arises due to amine groups in chitosan monomer that are protonated at acidic pH. DLS has been used to investigate the colloidal stability of nanoparticles in buffer solution of different pH. (Figure 3) All nanoparticles have good colloidal stability in phosphate buffer solution of pH 7.4 and bicarbonate buffer solution of pH 9.0 without any sign of precipitation within one month and without any change of particle size distribution. However, in acetate buffer solution of pH 4.5 nanoparticles shows partial precipitation at longer preservation which is also reflected in size distribution histogram. Fluorescence stability of colloidal nanoparticles has also been investigated in different buffer solution. (Supporting Information, Figure S8) Results show that fluorescence remains intact at different solution pH and for longer preservation. This result suggests that polydopamine coated nanoparticles can be preserved for months without any precipitation and with retained fluorescence. Robust nature of polydopamine coating has been investigated by exposing them under polar organic solvent where TPE is soluble. For example, nanoparticles with and without polydopamine coating are dispersed in methanol and result shows that polydopamine coated nanoparticles retain their fluorescence but without the polydopamine coating fluorescence is lost significantly. (Figure 5) Similar results are also observed using dimethylformamide and DMSO solvent. This results clearly show the robust nature of polydopamine coating and coated nanoparticle can be used as reliable fluorescent probe under complex biological condition. Another interesting aspect of this nanoparticle is the intact fluorescence in presence of conventional fluorescence quenchers. We have investigated the fluorescence stability of TPEbased nanoparticle in the presence of various fluorescence quenchers and compared with the widely studied quantum dot. We have used polyacrylate coated ZnS capped CdSe quantum


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dot for this comparative study, as this system is frequently used as bioimaging probe. Particles were dispersible in presence of tested quenchers. Results clearly show distinct advantage of TPE-based nanoparticle over quantum dot. (Figure 6) While quantum dot based nanoprobe quenches the fluorescence in presence of heavy metal ions or metal nanoparticle or in acidic pH; TPE-based nanoparticles retain their fluorescence under such environment. Galactose functionalized TPE-based nanoparticle as fluorescent cell imaging probe. In order to explore the bioimaging application potential, the specific cell labelling property of TPE@PD-gal and TPE-Chi@PD-gal has been investigated. Typically, cells are incubated with nanoparticle for one hour and washed cells are imaged under fluorescence mode with UV excitation. Result shows that TPE@PD-gal and TPE-Chi@PD-gal can selectively label HepG2 cells that have over-expressed galactose receptors. (Figure 7) Control experiments show that TPE@PD or TPE-Chi@PD cannot label HepG2 cells as they do not have galactose functionalization. Moreover, TPE@PD-gal or TPE-Chi@PD-gal cannot label HeLa cells that do not have over-expressed galactose receptor. (Supporting Information, Figure S9.) In another control experiment we found that aggregated TPE (without polydopamine coating) can level cells via non-specific interaction. (Supporting Information, Figure S10) These results also conclude that polydopamine coating is able to minimize the nonspecific binding issue of nanoparticle with cells and can be used for specific cell labelling applications. Cell viability study shows that nanoparticles are non-toxic even at high dose and with longer incubation time. (Supporting Information, Figure S11) These results also prove that galactose functionalized AIE-based nanoprobe can be synthesized, similar to galactose functionalized other nanoparticles.41-43

DISCUSSION TPE is a hydrophobic molecule, insoluble in water but soluble in polar organic solvents such as methanol/ethanol. In the water-ethanol mixture TPE aggregates, depending on the water



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volume %. In approach I we have adjusted the solvent composition for making colloidal aggregates and then trapped those aggregates via polydopamine coating. However, particles increase their size from 50-60 nm to 100-200 nm during the coating processes and this approach cannot produce particles of < 100 nm. The increase of particle size is reasonable considering the fact that no particle stabilizer is used and particles are free to grow larger during the early stage of coating. Although the use of surfactant stabilizer (Tween 20) can decrease the particle size to 20-30 nm, the polydopamine coating is restricted on their surface possibly due to restricted adsorption of dopamine. In approach II, we have used chitosan conjugated TPE that produces smaller particles of 20-30 nm and although particle grow during polydopamine coating, the final size remains < 100 nm. The smaller particle size of 20-30 nm is possibly due to self-assembly tendency of TPE-Chi that minimizes their uncontrolled aggregation. It is expected that hydrophobic TPE molecules assemble together, keeping hydrophilic chitosan and PEG outside that helps in forming smaller aggregates. Although < 50 nm particle size is ideal for biolabelling application and < 10 nm is required for urine excretion, such sizes are difficult to make using AIE molecules. This is mainly due to molecular aggregation requirement (that need 5-10 nm of minimum size) to attain reasonable fluorescence and coating of that nanoparticle that further increases the particle size. Polydopamine coating on the TPE/TPE-Chi aggregate surface occurs via adsorption of dopamine followed by oxidative polymerization reaction. Dopamine can adsorbed on the surface of TPE/TPE-Chi aggregate through π-π stacking of benzene rings, polymerize under mild basic condition and deposit on the surface of nanoparticle.28 Previously reported AIEbased nanoparticles are 50-400 nm in size with 8-30 % fluorescence quantum yield13,17,18 and these values are comparable with our observed values. In some selected cases 30-70 % fluorescence quantum yield is reported which is due to strong packing between AIEgens.44


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However, such type of strong packing is unexpected in present cases due to specific nature of TPE and used preparation condition. The relatively low fluorescence quantum yield of TPEChi based nanoparticle, as compared to TPE-based nanoparticle can be explained due to smaller size of nanoparticle with less compact packing between TPE units due to attached chitosan/PEG units. Previously reported AIE-based nanoparticles are synthesized by selfassembly of AIE molecule and using other polymer components such as amphiphilic block copolymers, BSA and chitosan.16 In those process AIE molecules are incorporated into the hydrophobic core of polymer micelle or covalently conjugated with polymers to make micelle in aqueous medium or noncovalently embedded into silica nanoparticles.17,18 These nanoparticles have very limited opportunity of post functionalization. In contrast presented nanoparticles are 50-200 nm in size and post functionalization can be easily achieved. Polydopamine based nanomaterials have wider range of application in biomedical field because of its unique bio-adhesion property. It is shown that quinone groups present in polydopamine favours adsorption of serum protein with preserved bioactivity and contribute to the enhancement of cell adhesion/proliferation.45 In addition, the cell adhesion property depends on cell type and reactive phenolic groups on polydopamine surface inhibits cell proliferation. Similarly, fibroblasts are shown to easily adhere and proliferate on polydopamine coated glass and megakaryocytic cell adhesion can be enhanced upon modification of the polydopamine film with hyaluronic acid.26,27 Moreover, polydopamine coated Au nanorod/polymer micelle/mesoporous silica nanoparticle are synthesized, functionalized with targeting biomolecules such as peptide/antibody/hyaluronic acid/folic acid and used for specific cell labelling via receptor mediated endocytosis.26-35 In the present case polydopamine coating offers 3 specific advantages. First, it allows TPEbased colloidal nanoparticle with intact fluorescence under complex bioenvironment. Second, the coating allows functionalization of nanoparticle using phenolic groups on their surface.



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Third, coating offers specific bio-labelling options with low non-specific binding. These advantages can be clearly observed by comparing the cell labelling performance with earlier reported TPE-based functional nanoparticle without any polydopamine coating.24 In absence of polydopamine coating, the TPE based nanoparticles non-specifically binds with cell surface and the fluorescence property of nanoparticle changes either due to change of selfassembly or due to cellular process. In contrast polydopamine coated TPE-nanoparticles do not have such non specific labelling and cell labelling is observed once they are functionalized with galactose.

CONCLUSION We have demonstrated that polydopamine coating can be adapted in deriving AIE molecule based functional nanoparticle. We have successfully synthesized galactose functionalized colloidal nanoparticle of 50-200 nm size with 6-12 % fluorescence quantum yield and used to label cells with over-expressed galactose receptors. These nanoprobes are colloidally stable under physiological condition and their fluorescence remains intact under complex biological condition. Polydopamine coating approach may be extended for making AIE-molecule-based other nanoprobes towards various biomedical applications. ASSOCIATED CONTENT Supporting Information Details of control experiments in making nanoparticle and related data, mass spectral characterization of thiolated galactose, galactose estimation in nanoparticle, additional characterization data on polydopamine coating, colloidal stability data of polydopamine coated nanoparticle and control cell labeling data. This material is available free of charge via the Internet at http://pubs.acs.org. Notes 16 ACS Paragon Plus Environment

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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. Properties of polydopamine coated nanoparticles prepared from TPE-based AIE molecule. Method

Nanoparticle abbreviation

Hydrodynamic size

Approach I Approach I

TPE@PD TPE@PD-gal

100-200 nm 100-200 nm

Zeta potential (mV) at pH 4.5 7.4 9.0 -1 -13 -16 -2 -7 -12

Approach II Approach II

TPE-Chi@PD TPEChi@PD-gal

50-100 nm 50-100 nm

6 3

-2 -4

-8 -6

Fluorescence quantum yield 10-12 % 10-12 % 6-8 % 6-8 %

Cell labelling application

no labelling specific labelling no labelling specific labelling



Page 24 of 33

Scheme 1. Synthesis strategy of TPE-based nanoparticle by approach I (a) and approach II (b). In approach I, chloroform solution of TPE is injected into 50 volume % water-ethanol mixture under stirring and aggregated TPE is formed with the evaporation of chloroform. Next, dopamine solution is added under mild alkaline condition to initiate the polydopamine coating around aggregated TPE. In approach II, chitosan and PEG conjugated TPE is aggregated under methanol-water mixture and then polydopamine coating is initiated by adding dopamine under mild alkaline condition.


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Scheme 2. a) Synthesis scheme in making thiolated galactose from lactose. b) Schematic representation of galactose functionalization of polydopamine coated nanoparticle prepared from TPE/TPE-Chi.



a)

Water % 100 90 80 70 60 50 40 30 20 10 0

Fluorescence intensity (a.u.)

50000 40000 30000 20000 10000 0 450

500

550

600

650

700

Wavelength (nm)

b) Fluorescence Intensity (a.u.)

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 33

Water %

1000

100 90 80 70 60 50 40 30 20 10 0

800 600 400 200 0 400

450

500

550

600

650

700

Wavelength (nm)

Figure 1. Demonstration of aggregation induced emission property of TPE (a) and TPE-Chi (b), showing that fluorescence increases in a methanol-water medium with the increasing water %. The fluorescence spectra are measured with 390 nm excitation (for TPE) or 370 nm excitation (for TPE-Chi).


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Figure 2. Absorption and fluorescence spectra of TPE-based nanoparticles and TPE-Chibased nanoparticles. a) TPE nanoparticle without polydopamine coating (i), polydopamine coated TPE nanoparticle, TPE@PD (ii), galactose functionalized TPE@PD, TPE@PD-gal (iii). b) TPE-Chi nanoparticle without polydopamine coating (i), polydopamine coated TPEChi nanoparticle, TPE-Chi@PD (ii), galactose functionalized TPE-Chi@PD, TPE-Chi@PDgal (iii). Excitations of 390 nm and 370 nm are used for TPE and TPE-Chi based nanoparticles, respectively. Inset picture shows digital images of respective nanoparticles under day light (left) or UV light. 27 ACS Paragon Plus Environment


a)

b) 20

0.4

10

10 0 0 30

pH 9 0.4

10

0.0 0.1 10 10 3 105 Time (µs)

200 400 Size (nm)

0 0

600 d)

20

Correlation coefficient

0 30

20 10

0.8 0.4

0.8 0.8


10 0 0

pH 7.4

0.6

0.4 0.4 0.2

0.0 0.0 0.1 10 10 3 105 Time (µs)

0.8

pH 9

0.0 0.1 10 10 3 105 Time (µs)

200 400 Size (nm)

600


0.6

0.40.4 0.2

0.00.0 0.1 10 103 105 Time (µs)

pH 9

0.8 0.4

0.0 0.1 10 103 105 Time (µs)

200 400 Size (nm)

600 0.9

0.8

0.8

pH 4.5

0.7

0.6

0.5

0.4

0.4

0.3

0.2

0.1

0.0 0.1 10 103 105 Time (µs)

0

20 10

0.8

1.0

0.9

0.7

0.4

0.6

0.5

0.4

0.3

0.2

0.0 0.1 10 103 105 Time (µs) 0.0

0.8 0.8

0.9

0.8

10 0 0

pH 7.4

0.8

0.1

0 30

20

0.4

pH 7.4

0.80.8

0.0

0.0 0.1 10 10 3 105 Time (µs)

0 30 20

10

Number (%)


10

0

20

0.8

pH 4.5

20

10


0.0 0.1 10 10003 100000 5 0.1 10 10 10 Time (µs)

20

pH 4.5

0.4

0 30


0.4

Number (%)

0.8

0 20

c)


10


Number (%)

pH 7.4

0.8

0.0 0.1 10 103 105 Time (µs)


0.0 0.1 10 10 3 105 Time (µs)

0 20


0.80.8


10

pH 4.5 Correlation coefficient

20

Number (%)

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 28 of 33

pH 9

0.7

0.6

0.6

0.5

0.4 0.4

0.4

0.3

0.2

0.2

0.1

0.0 0.0 0.1 10 103 105 Time (µs) 0.0

200 400 Size (nm)

600

Figure 3. a) Comparative size distribution histogram of a) TPE@PD b) TPE@PD-gal c) TPEChi@PD d) TPE-Chi@PD-gal in acetate buffer of pH 4.5, in PBS buffer of pH 7.4 and in bicarbonate buffer of pH 9.0.


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Figure 4. SEM images of a). TPE nanoparticle without polydopamine coating (i), polydopamine coated TPE nanoparticle, TPE@PD (ii), galactose functionalized TPE@PD, TPE@PD-gal (iii). b) TPE-Chi nanoparticle without polydopamine coating (i), polydopamine coated TPE-Chi nanoparticle, TPE-Chi@PD (ii), galactose functionalized TPE-Chi@PD, TPE-Chi@PD-gal (iii).



1000

Fluorescence Intensity (a.u.)

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 30 of 33

water methanol

800 600 400 200 0

Figure 5. Evidence of robust coating by polydopamine that inhibits disassembly of AIE in methanol. Nanoparticles without any polydopamine coating lose their emission in methanol but polydopamine coated particles retain their fluorescence.


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Au

600

Zn2+

800

Control (pH 7.4)

0

Fe2+

Cu2+

200

Fe3+

Ag

400

pH 4.5


1000

pH 9

a)

Fluorescence Quencher

b)

600

Au

Ag

Zn2+

Fe2+

Fe3+

Cu2+

pH 9

pH 4.5

800

Control (pH 7.4)


1000

400 200 0


Au Ag

Zn2+

Fe2+

Fe3+ Cu2+

600

pH 9

800

pH 4.5

1000

Control (pH 7.4)

c)


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


400 200 0


Figure 6. a) Comparative fluorescence stability of quantum dot (a), TPE@PD (b) and TPEChi@PD (c) in presence of different fluorescence quenchers. Final concentration of metal ions is kept at 0.1 mM and concentration of silver and gold of respective nanoparticles solution are maintain at 0.5 mM. Other conditions are mentioned in the experimental section. 31 ACS Paragon Plus Environment


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Figure 7. Bight field (BF) and fluorescence (F) image of HepG2 cells labeled with TPE@PD (i), TPE@PD-gal (ii), b) TPE-Chi@PD (iii) and TPE-Chi@PD-gal (iv) showing that galactose functionalization offers selective labeling of HepG2 cells. Typically, cells are incubated with colloidal nanoparticle for one hour and washed cells are imaged under BF and F mode under UV excitation. Scale bar represent 50 microns. 32 ACS Paragon Plus Environment

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