Carbon Dots-Stimulated Amplification of Aggregation-Induced

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Carbon Dots Stimulated Amplification of Aggregation Induced Emission of Size Tunable Organic Nanoparticles Pritam Choudhury, and Prasanta Kumar Das Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01631 • Publication Date (Web): 22 Jul 2019 Downloaded from pubs.acs.org on July 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 48 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

Langmuir

Carbon Dots Stimulated Amplification of Aggregation Induced Emission of Size Tunable Organic Nanoparticles Pritam Choudhury and Prasanta Kumar Das* School of Biological Sciences, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700032, India.

*To whom correspondence should be addressed: [email protected]

1 ACS Paragon Plus Environment

Langmuir 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

ABSTRACT Carbon dots (CDs) induced microstructural modulation and amplification of emission intensity of self-aggregated fluorescent organic nanoparticles (FONPs) is a challenging task since CD is well known fluorescence quencher. In the present study, we have designed Ltyrosine tagged hydrophobically (C-10) tailored naphthalene diimide derivative (NDI-i), which formed FONPs in THF-water binary solvent mixture. NDI-i exhibited aggregation induced orange emission (AIE) at 580 nm up to fw = 70 vol% of water in THF via excimer formation in combination with intramolecular charge transfer (ICT) upon excitation at 350 nm. Beyond fw = 70 vol%, the emission intensity gradually reduced up to fw = 99 vol% due to poor water dispersibility of NDI-i FONPs. Doping of hydrophobically (C-2 to C-11 alkyl chain) surface functionalized CDs (CD-i-iii) within self-aggregates of NDI-i FONP at fw = 99 vol% resulted in the modulation of both morphology and emission intensity of resulting selfassembled nanoconjugate. In presence of C-2 alkyl chain tethered CD, the emission intensity of FONP-CD nanohybrid got quenched than that of native NDI-i FONP. The emission intensity of NDI-i FONP markedly enhanced by 3.6-5.0 fold upon inclusion of C-6 and C-11 alkyl chain containing CDs. Increasing the alkyl chain length on CD surface facilitated the inter-chain hydrophobic interaction between the organic nanoparticles and surface functionalized CDs to form larger size CD-doped fused FONPs. The extent of ICT between -donor and -acceptor residue became more efficient to exhibit enhanced AIE due to the accumulation of more NDI-i around CD surface through inter-chain hydrophobic interaction. The C-11 alkyl chain containing CD integrated FONPs showed the brightest orange emission with superior aqueous stability. This water dispersible, orange emitting, cytocompatible NDIi-CD-iii FONPs were explored for long-term bioimaging of mammalian cells.


Page 2 of 48

Page 3 of 48 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

Langmuir

INTRODUCTION The discovery of organic luminogens with aggregation induced emission (AIE) properties leads to the development of fluorescent organic nanoparticles (FONPs).1-6 These FONPs are finding immense significance in diverse field ranging from electronic to photonic, conducting materials to sensors, medicine to biotechnology, and so forth.7-13 The AIE luminogens (AIEgens) in benign solvents (e.g., THF, DMSO, DMF etc.) are mixed with their poor solvent (water) to develop FONPs.14 Numerous approaches have been acquired to develop stable and strongly emitting AIE-gens in biological domain through surface modifications (e.g., hydrophilic polymer encapsulation, pegylation or silica coating) of the FONPs.12,15-18 However, in many instances, such polymer or silica coated organic nanoparticles often show detrimental effect like oxidative stress, inflammatory response toward biological system.19,20 Hence, addition of some exogenous non-toxic surface modifier or stabilizers is on high demand for the amplification of AIE by altering the physicochemical properties of FONPs. Over the past decade, amalgamation of carbon nanomaterials (CNMs) with diverse soft-materials has been explored mostly by exploiting the distinctive surface chemistry of either of the components.21-26 Inclusion of CNMs to the self-assembled structures aided superior changes in their physicochemical properties with the assistance of several noncovalent interactions such as - stacking, hydrogen bonding, van der Waals forces.21,22,27,28 Despite the growing interest in carbon nanotubes/graphene/graphene oxide based softnanocomposites, development of carbon dot (CD) integrated soft-nanoconjugate has not been widely explored yet.29-31 CDs, ‘zero dimensional’ allotrope of carbon, have had a tremendous impact in scientific research and technological progress due to their versatile surface passivation, chemical robustness, unique photoluminescence, high water solubility, and low cytotoxicity.32,33 The influence of surface functionalized CDs to tune the microstructure as well as stability of the FONPs is an unresolved issue of fundamental importance. 3 ACS Paragon Plus Environment

Langmuir 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 4 of 48

Amplification in the emission intensity of the AIE-gens in self-aggregated state upon interaction with integrated CDs is a challenging task considering the distinctive emissive property and fluorescence quenching property of CDs. With this aim, in the present study we have amalgamated hydrophobically tailored naphthalene diimide (NDI-i, Figure 1a) based FONPs with surface functionalized (C-2 to C11 alkyl chain) CDs (CD-i-iii, Figure 1a) to construct FONP-CD nano-conjugates with amplified AIE. The synthesized native NDI-i showed very weak AIE (orange, em = 580 nm) via excimer formation in tetrahydrofuran (THF)-water (1:99, v/v) upon excitation at 350 nm. In presence of CD-i (C-2 alkyl chain containing), this feeble emission intensity of the FONP solution got further reduced. Upon addition of higher alkyl chain (C-6, CD-ii and C-11, CDiii) tethered CDs, the FONP-CD nanoconjugate showed bright orange emission, whose intensity is notably higher than that of native NDI-i FONP. The increment of the alkyl chain length on the CD surface efficiently enhanced the emission intensity of the FONP-CD nanohybrid. Moreover, hydrophobic modulation of alkyl chain on the CD surface could also regulate the dimension of the organic nanoparticles through favourable inter-droplet interaction. To the best of our knowledge, this is the first ever approach to amplify the AIE of FONPs by modulating the microstructure in presence of carbon dots. Furthermore, long term cellular imaging of cancer cell was carried out using CD-iii doped FONPs owing to its superior emission intensity, water dispersibility and cytocompatibility. EXPERIMENTAL SECTION Materials. 1,4,5,8-Naphthalenetetracarboxylic dianhydride, 1-naphthaleneethanol, thiazolyl blue tetrazolium bromide (MTT), 8-anilino-1-naphthalenesulfonic acid (ANS), methyl viologen dichloride hydrate, dialysis bag (MWCO 2000) and all deuterated solvents were purchased

from

aminoundecanoic

Sigma-Aldrich. acid,

L-tyrosine,

glycine,

di-tert-butyldicarbonate 4 ACS Paragon Plus Environment

6-aminocaproic (Boc-anhydride),

acid,

11N,N-

Page 5 of 48 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

Langmuir

dicyclohexylcarbodiimide

(DCC),

4-N,N-(dimethylamino)

pyridine

(DMAP),

N-

hydroxybenzotriazole (HOBT), trifluoroacetic acid (TFA), solvents, silica gel (60-120 mesh and 100-200 mesh), and all other reagents were purchased from SRL, India. We have used Milli-Q water throughout the experiments. Dulbecco’s Modified Eagle’s Medium (DMEM), heat activated fatal bovine serum (FBS), trypsin from procine pancreas were purchased from Hi-Media, India. NMR spectra were recorded in an AVANCE 300/400 MHz (Bruker) spectrometer. MALDI-TOF spectra were performed on Bruker Ultraflex MALDI mass spectrometer with 2,5-dihydroxy benzoic (DHB) acid as matrix. Biobase BK-FD12P freezedrier was used for lyophilization and centrifugation was performed in Thermo Scientific Espresso centrifuge machine. Synthesis of NDI-i. The complete synthetic procedure is described in Scheme S1 (Supporting Information). At first, Boc-protected methyl ester of L-tyrosine was substituted by n-decyl group (-C10H21) to get tri-functionalized L-tyrosine using 1-bromodecane (1.5 equiv) and Na2CO3 (1.5 equiv). The reaction was carried out in dry DMF and stirred at 80 °C for 24 h. DMF was removed under vacuum distillation and DCM was added to extract the crude mass. The crude mass was purified using 60-120 mesh silica gel (eluent: 1% methanol in chloroform) to obtain C-10 alkylated L-tyrosine at the -OH terminal. The purified mass was then dissolved in MeOH and the reaction mixture was stirred at room temperature in presence of aqueous NaOH solution (1.1 equiv). The resulting reaction mixture was concentrated and then acidified with 1(N) HCl solution and the produced carboxylic acid was extracted with EtOAc. Next, the compound (1.2 equiv) was coupled with 1naphthaleneethanol (1 equiv) in presence of DCC (1.1 equiv), DMAP (1.1 equiv) and HOBT (1.1 equiv). The coupling reaction was carried out under N2 atmosphere in dry DCM for 12 h. The organic layer was washed with 1(N) HCl and then concentrated to obtain the crude mass. The coupling product was purified by column chromatography (eluent: 1% methanol in 5 ACS Paragon Plus Environment

Langmuir 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

chloroform). A reported protocol was followed to obtain the free amine derivative by the deprotection of Boc-group from the coupling product upon treated with TFA (1.5 equiv).6 Finally, a condensation reaction was performed in dry DMF under heating condition (90 °C, 12 h, N2 atm.) between 1,4,5,8-naphthalenetetracarboxylic dianhydride (1.0 equiv) and free amine derivative (2.2 equiv) to obtain NDI-i. The target product (NDI-i) was purified by column chromatography using 100-200 mesh silica gel (eluent: 2% MeOH/CHCl3). Characterization data (1H/13C-NMR and MALDI-TOF mass spectrometry) for NDI-i has been provided in the Supporting Information. Synthesis of Surface Functionalized Carbon Dots. Surface functionalized carbon dots (CD-i-iii) were synthesized by following the reported literature protocol.34 To synthesize CDi, first sodium salt of glycine was obtained by the mixing of glycine (14 mmol) with an equivalent amount of NaOH solution (2 mL). To this, aqueous citric acid solution (14 mmol, 2 mL) was added by to maintain the molar ratio 1:1. This mixture was evaporated to dryness at 100 °C. The sticky mass was dried at 80 °C for72 h. the crushed solid fine powder was then heated in a furnace at 200 °C for 2 h in a porcelain crucible and then cooled to room temperature. The brownish-black product was dissolved in 25 mL of hot water. Then aqueous part was centrifuged at 12,000 rpm for 30 min to remove the insoluble pellet. The supernatant was transferred to dialysis bag (MWCO 2000) and the dialysis process was carried out against Milli-Q water. The resulting solution was lyophilized to get the corresponding carbon dots (CD-i). Similar method was applied for the synthesis of CD-ii and CD-iii. All the synthesized CDs were characterized by 1H-NMR study (see Supporting Information). Preparation of Organic Nanoparticles. NDI-i was soluble in THF. Hence, stock solution of NDI-i (5 mM) was prepared in THF. From this stock solution, required amount of aliquot was added to the THF-water mixture of different ratio to attain concentration 20 M for different experiments. Similarly, from the stock solution (in water) of CDs (5 mg/mL) 6 ACS Paragon Plus Environment

Page 6 of 48

Page 7 of 48 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

Langmuir

required volume of sample was mixed with the NDI-i (20 M) solution prepared in fw = 99 vol% and different microscopic as well as spectroscopic studies were investigated for the characterization of the soft-nanohybrids. Transmission Electron Microscopy (TEM) Study. Transmission electron microscopy was performed to monitor the self-assembled structure of NDI-i (20 M) in fw = 99 vol%. 4 L of aliquot was drop cast on a 300 mesh Cu grid (carbon coated) and dried to obtain TEM images. Similar techniques were applied to obtain the TEM images of CDs and CD doped self-assembled systems. For, CD doped self-assembled systems the concentration of each CD was varied from 0.5 g/mL to 5 g/mL keeping the concentration of NDI-i fixed at 20 M. To monitor negatively stained TEM images, 1 L of freshly prepared uranyl acetate (1% w/v) was used. TEM study was also performed in case of CD-i doped NDI-i organic nanoparticles at pH = 2.0 (20 mM KCl-HCl buffer). The samples were kept under vacuum for 4 h before imaging. JEOL JEM 2100F UHR microscope was used for TEM experiments. Dynamic Light-Scattering (DLS) Study. Mean hydrodynamic diameter (Dh) of organic nanoparticles derived from NDI-i (20 μM), surface functionalized CDs and CD doped (varying concentration from 0.5 g/mL-5 g/mL) NDI-i in fw = 99 vol% were determined by Zen 3690 Zetasizer Nano ZS instrument (Malvern Instrument Ltd.). DLS study was also performed in case of CD-i doped NDI-i organic nanoparticles at pH = 2.0 (20 mM KCl-HCl buffer). The scattering intensity was monitored at 175° angle and data were analyzed in Malvern Zetasizer software. UV-Visible Study. UV-visible spectra of NDI-i (20 μM) in different solvent compositions of THF-water ( fw = 0-99 vol%) was monitored in Perkin Elmer Lambda 25 spectrophotometer for the investigation of self-aggregation pattern. UV-visible study for NDI-i (20 μM) was also carried out in different organic solvents (benzene, toluene, o-xylene, chloroform, dichloromethane) with increasing polarity. 7 ACS Paragon Plus Environment

Langmuir 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

1H-NMR

Study. 1H-NMR spectra of NDI-i (1 mM) was recorded in Avance 300 MHz

(Bruker) spectrometer in DMSO-d6 and different solvent mixture of DMSO-d6-D2O (3:1, v/v and 1:3, v/v) to investigate the non-covalent interactions during self-assembly. Photoluminescence Study. Emission spectra of NDI-i (20 M) in THF-water binary solvent mixture of different solvent ratios (fw = 0-99 vol%) were monitored in a Varian Cary Eclipse luminescence spectrometer at ex = 350 nm. Photoluminescence study for NDI-i (20 M) was also carried out in different polarity organic solvents (benzene, toluene, o-xylene, chloroform, dichloromethane) for the investigation of intramolecular charge-transfer (ICT) phenomenon. Excitation-dependent emission spectra of aqueous solution of CD-i-iii (5 g/mL of each) were also monitored with excitation range of 300-400 nm. The emission spectra of free ANS and ANS doped self-assembled systems (NDI-i FONPs and CD doped NDI-i FONPs) was also recorded. In each case, the concentration of the ANS was maintained at 10 M. The photoluminescence property of NDI-i (20 M) solution upon incubation of different surface functionalized carbon dots (CD-i/ii/iii) was investigated by varying the concentration of CDs from 10 ng/mL to 5 g/mL. The fluorescence spectra of NDI-i FONPs ([NDI-i] = 20 M) and NDI-i-CD-iii FONPs ([NDI-i] = 20 M, [CD-iii] = 0.5 g/mL) was monitored in presence of methyl viologen dichloride hydrate (MV). The concentration of MV was varied from 5 M to 20 M. Time-Resolved Study. Picosecond diode laser IBH-405 was used to determine time correlation single photon count (TCSPC) measurement. NDI-i solutions prepared in THF and (1:99, v/v) THF-water were excited (ex) at 375 nm and monitored the emission (mon) at 400 nm (in THF) and at 580 nm (in 1:99, v/v THF-water). Similarly, the emission of surface functionalized CD (CD-i/ii/iii) doped NDI-i solution at fw = 99 vol% was also recorded (ex = 375 nm and mon = 580 nm). IBH DAS6 software was utilized for analyzing fluorescence 8 ACS Paragon Plus Environment

Page 8 of 48

Page 9 of 48 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

Langmuir

decays. Experimental time resolved fluorescence decay p(t) was analyzed by following equation (1).

( ) 𝑡

𝑛

𝑝(𝑡) = 𝑏 + ∑𝑖 𝛼𝑖 exp ― 𝜏𝑖

(1)

Where, n = number of discrete emissive species, b = baseline correction (“dc” offset), and i = pre-exponential factors and i = excited state fluorescence lifetimes associated with the ith component, respectively. For multi-exponential decays, equation (2) was used to calculate the average life-time. 𝑛

=

∑𝛼 𝜏

𝑖 𝑖

𝑖=1

Where 𝛼𝑖 =

𝛼𝑖 ∑𝛼𝑖

(2)

which indicates the contribution of a decay component.

Quantum Yield (QY) Estimation. QY of NDI-i (u) was calculated by using equation 3, with respect to a reference fluorophore of known QY (s) , ∅𝑢 =

( ) 𝐴𝑠𝐹𝑢𝑛2𝑢 𝐴𝑢𝐹𝑠𝑛2𝑠

∅𝑠

(3)

Here, Au = 348 nm and As = 384 nm are absorbance of unknown and reference sample at respective ex. Fu and Fs are integrated fluorescence intensity for the unknown and known sample excited at respective ex. nu and ns are refractive indices of solvents in which the unknown sample and known sample was prepared, respectively. Solutions having similar absorbance (< 0.01) were used for estimation of QY. Herein, quinine sulfate in 0.1 M H2SO4 was considered as reference (QY (s) = 54.0%). Similarly, QYs of CD doped NDI-i was also estimated in fw = 99 vol% by following the same protocol. Stability Experiment of NDI-i and CD Doped NDI-i FONPs. Stability of NDI-i FONPs (20 M) in fw = 99 vol% was monitored for a long time period (up to 7 days). The absorbance 9 ACS Paragon Plus Environment

Langmuir 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 10 of 48

and the emission intensity of this solution were recorded at 384 nm and 580 nm, respectively at different duration of time for the determination of suspension stability of NDI-i FONPs. Similar protocol was performed to determine the stability of CD-ii and CD-iii doped NDI-i FONPs (20 M) at fw = 99 vol% by varying concentration of CDs. % stability was calculated by measuring the relative absorbance as well as relative emission intensity at different time scale. MTT Assay. Cytocompatibility of NDI-i FONPs as well as CD-doped NDI-i FONPs (NDI-iCD-iii) at fw = 99 vol% was investigated by MTT assay. In this study, quantity of produced formazan upon reduction was calculated spectrophotometrically by solubilizing it in DMSO. Amount of produced formazan is proportional to number of live cells. B16F10 cells were cultured (2 × 104 cells per well) in a 96-well plate for 18-24 h before the assay. MTT assay with NDI-i FONPs (fw = 99 vol%) was investigated over a concentration range of 5-40 M in a microtitre plate. B16F10 cells were incubated with NDI-i for 24 h (37 °C, 5% CO2 atmosphere). 10 L from MTT stock solution (5 mg/mL) in PBS was added to the above mixture and the cells were incubated for another 4 h. Precipitated formazan was dissolved in DMSO, and absorbance was recorded at 570 nm using BioTek1 Elisa Reader. Similarly, cytotoxicity of NDI-i-CD-iii ([NDI-i] = 20 M) was also investigated by varying the concentration of CD-iii (1-5 g/mL). Number of live cells was expressed as % viability by following the equation: A570(Treated Cells) ― Background

% viability = A570(Untreated cells) ―

Background

× 100

(4)

Cellular Imaging. B16F10 cells were grown (104 cells/well) within a chamber slide for 1824 h. Cells were treated with NDI-i (20 M) and NDI-i-CD-iii ([NDI-i] = 20 M and [CD-iii] = 5 g/mL) FONPs in fw = 99 vol% for different time scale (6-24 h). The incubated cells were washed with PBS buffer and fixed with paraformaldehyde (4%) for 30 min. Glycerol 10 ACS Paragon Plus Environment

Page 11 of 48 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

Langmuir

(50%) was used to mount the cells on slide, which was covered using cover slip and kept for 24 h. Cellular imaging was carried out in Olympus IX-83 microscope (excitation filter: BP 530-550 nm and band absorbance filter covering below 570 nm for red fluorescence image) at 40× magnification. Flow Cytometry. B16F10 cells were treated with NDI-i ([NDI-i] = 20 M) and NDI-i-CD-iii ([NDI-i] = 20 M and [CD-iii] = 5 g/mL) FONPs for different time period (6-24 h). After (6-24 h) of incubation, the cells were washed several times with PBS buffer. The treated cells were then trypsinized, centrifuged and suspended in 500 μL of PBS. BD Accuri C6 flow cytometer was used to analyze the treated cells at emission wavelength using a 585±40 nm (FL-2) bandpass filter. Initially, 5 × 104 cells were taken for seeding and data was collected from 104 cells. RESULTS AND DISCUSSION Molecular Designing of NDI-i. Molecular design plays a crucial role to show strong emission of the luminophore in the self-assembled state. Restriction of intramolecular rotation (RIR) of the luminophore during self-assembly is one of the key factors to facilitate the AIE by reducing the non-radiative decay.2 Moreover, higher emission wavelength is always beneficial for cellular imaging because low optical waves can easily penetrate tissue and minimize background auto-fluorescence.35 Insertion of donor-acceptor (D-A) residue in the molecular framework of the amphiphilic compound can influence the emission at higher wavelength through strong intramolecular charge transfer.36 Naphthalene diimide (NDI), an electron deficient n-type organic semiconductor of ‘rylene’ family, has been widely used to develop light harvesting triad, fluorescent sensors, biological probes and so on.36-41,6,42-45 In this context, we have designed NDI-i (Figure 1a and Scheme S1, Supporting Information) by choosing NDI (-acceptor) as electron deficient conjugated hydrophobic core which was tagged with -electron rich naphthyl residue (-donor). L-tyrosine residue acted as an anchor 11 ACS Paragon Plus Environment

Langmuir 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

between the donor-acceptor residues. Moreover, chirality of the L-tyrosine residue may induce non-planarity in the molecular backbone which may prompt the RIR during the selfaggregation. Intermolecular interaction between the aromatic rings present in NDI-i may facilitate the self-assembly process.6,36 Additionally, the long n-decyl (-C10H21) alkyl chain substitution at the both terminal is expected to undergo strong inter-chain hydrophobic interaction, which can diminish aggregation caused quenching (ACQ) via strong - interaction. This synthesized NDI derivative was characterized by 1H-/13C-NMR and MALDI-TOF mass spectrometry (see Supporting Information). Self-assembly of NDI-i: Microscopic and Spectroscopic Investigation. The synthesized NDI derivative was soluble in THF. At and above 30% water content in the THF solution of NDI-i, change in physical appearance of the solution from transparent to translucent indicated the initiation of self-assembly by NDI-i (Figure 2a). Increase in the water content gradually enhanced the translucency of the solution up to 70 vol% water content. However, a marked drop in the translucency was noted at 90 vol% and 99 vol% water content presumably due to the lower solubility of NDI-i organic nanoparticles at higher water content. To elucidate the morphology of the self-assembled structure, we have monitored the TEM image of NDI-i (20 M) at fw = 99 vol% (Figure 1b and S1a, Supporting Information). Spherical aggregates of diameter in the range of 30-40 nm with no clear contrast were observed in the TEM image indicating the solid nature of the self-assembled structures (Figure 1b and S1a, Supporting Information). This observation confirmed the successful formation of organic nanoparticles by NDI-i. The size of NDI-i organic nanoparticles was further investigated from DLS study. The Dh of the spherical aggregates was found to be in the range of 25-45 nm at the same concentration that was used in TEM study (Figure S1b, Supporting Information). The self-aggregation pattern of the organic nanoparticles was investigated by UVvisible study. In pure THF, NDI-i (20 M) showed characteristic UV-band in the range 33012 ACS Paragon Plus Environment

Page 12 of 48

Page 13 of 48 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

Langmuir

400 nm with max at 379 nm because of - transition polarized along the long axis of NDI chromophore (Figure S2, Supporting Information).46 With gradual increase of water in THF, the absorption maxima gradually red shifted to higher wavelength. At (1:99, v/v) THF-water, the max was observed at 384 nm (Figure S2, Supporting Information). This bathochromic shift (5 nm) of max delineates the formation of self-aggregated species through Jaggregation. Involvement of different type of non-covalent interaction e.g., - stacking, hydrophobic interaction during self-assembly was investigated by solvent dependent 1HNMR study. In DMSO-d6, characteristic NMR signals for aromatic protons of NDI-i appeared at  = 8.24 ppm (NDI core) and in the range of  = 6.59-8.10 ppm (benzene unit Ltyrosine and naphthyl group) (Figure S3a, Supporting Information). These  values gradually upfield shifted and simultaneously the signal intensity also got reduced upon addition of selfaggregating solvent, D2O. At (1:1, v/v) DMSO-d6-D2O, the  was found with reduced intensity at 7.84 ppm and in the range of 6.20-7.71 ppm for the respective aromatic protons (Figure S3a, Supporting Information). This result established the involvement of intermolecular - interaction between aromatic residues during self-assembly. In addition, the NMR signals for methyl and methylene protons of the long alkyl chain (-C10H21) also got upfield shifted from  = 0.82-1.68 ppm (DMSO-d6) to  = 0.72-1.55 ppm (1:1 v/v, DMSOd6-D2O) with very poor intensity (Figure S3b, Supporting Information). This observation reiterates that upon addition of D2O, inter-chain (alkyl chain) hydrophobic interaction between the alkyl residues got increased. All the microscopic and spectroscopic evidences confirmed the formation of spherical organic nanoparticles with the assistance of various non-covalent interactions. Investigation of AIE of NDI-i Organic Nanoparticles. After successful formation of organic nanoparticles, we have monitored the emission of NDI-i (20 M) both in molecularly 13 ACS Paragon Plus Environment

Langmuir 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 14 of 48

dissolved state (THF) as well as in the self-assembled state. THF solution of NDI-i showed very weak blue emission upon UV-light exposure (ex = 365 nm) (Figure 2b). At 30% water content, the emission colour subsequently changed to feeble orange when irradiated with UVlight of same excitation wavelength (Figure 2b). Emission intensity of the NDI-i solution gradually enhanced up to fw = 70 vol% (Figure 2b). However, this orange emission was found to be reduced progressively beyond fw = 70 vol% possibly due to poor water dispersibility of hydrophobically tailored NDI-i molecules. This has also been reflected when comparatively less translucent NDI-i at fw = 99 vol% showed weaker orange emission than that of at fw = 70 vol% upon excitation with same UV-light (Figure 2b). This

characteristic

emission

behaviour

provoked

us

to

examine

the

photoluminescence property of the NDI-i in different fraction of THF-water. In THF solution of NDI-i, the emission maximum (em) was appeared at 404 nm at an excitation (ex) of 350 nm (Figure 3a). Upon addition of water (up to fw = 20 vol%), the emission intensity was found to be decreased. At 30% water content, the monomeric emission band almost disappeared and a new emission band (excimer) at 580 nm was observed (Figure 3a). The emission intensity of this newly generated excimer band gradually enhanced up to fw = 70 vol% (~1.6-fold enhancement in intensity than that was found for the molecularly dissolved monomeric form of NDI-i in THF, Figure 3a, b). This phenomenon suggested that NDI-i exhibited higher emission intensity at 580 nm via excimer formation due to AIE. However, the excimer band intensity consecutively decreased with further addition of water beyond fw = 70 vol%, which is in concurrence with the observed weak orange emission of suspended FONP solution upon UV-light exposure in the preceding paragraph (Figure 2b, 3a). At 99 vol% water content, the emission intensity of the excimer band intensity reduced to almost 60% with respect to the intensity observed at fw = 70 vol% water content (Figure 3a, b).


Page 15 of 48 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

Langmuir

To further elucidate the AIE behaviour of NDI-i, time resolved study was performed with picosecond excitation at 375 nm of NDI-i in THF and (1:99, v/v) THF-water (Figure 3c). In each case, tri-exponentially fitted decay curves were obtained. For THF solution of NDI-i, the emission was monitored at λmon = 404 nm and the average life-time value () was 0.04 ns (Table 1). At fw = 99 vol%, the value was 0.81 ns (λmon = 580 nm) (Table 1). This long decay lifetime possibly arises from pre-associated chromophores in higher percentage of water. Thus, TCSPC experiment further confirms that the NDI-i exhibited AIE of through pre-associated excimer formation. In addition, influence of charge transfer process to the AIE phenomenon was also examined by monitoring the UV-visible and emission spectra of NDI-i at various organic solvents of different polarities (Figure 3d, e). No significant changes in the absorbance maxima were found by changing the solvent from non-polar benzene to highly polar dichloromethane (Table 2, Figure 3d). However, the emission maximum gradually red shifted with increasing solvent polarity (Table 2, Figure 3d). The Stokes shift () of NDI-i in different solvents was estimated and subsequently, the solvatochromic property was also quantitatively described by the Lippert-Mataga equation 5:47,48 ∆𝜈 = 𝜈𝑎 ― 𝜈𝑒 =

2∆𝑓 ℎ𝑐𝑎

3

(𝜇𝐸 ― 𝜇𝐺)2 + 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡

(5)

Where, νa = maximum absorbance wavenumber, νe = maximum emission wavenumber, μG = ground state dipole moment, μE = excited state dipole moment, h = Planck’s constant, c = speed of light, a = Onsager solvent cavity and n = refractive index of the respective solvent. The orientational polarizability or solvent polarity parameter Δf, defined in equation 6, is the measure of solvent polarity. 𝜀―1 𝑛2 ― 1 ∆𝑓 = ― 2𝜀 + 1 2𝑛2 + 1

(6) 15 ACS Paragon Plus Environment

Langmuir 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

Where, ε = static dielectric constant and n = optical refractive index of the solvent. Linearity between  vs Δf and positive solvatochroism of NDI-i delineates the intramolecular charge transfer (ICT) between the donor (naphthyl unit) and the acceptor (NDI core) residue (Figure 3f). This ICT process has the influence in red shifting the emission maxima during AIE phenomenon. Inclusion of Surface Functionalized Carbon Dots in NDI-i FONP Solution. This hydrophobically tailored NDI-i FONPs showed AIE but the emission intensity notably dropped at higher percentage water content possibly due to its poor water dispersibility. To improve the prospects of the synthesized NDI-i FONPs in biomedicine/bioimaging, it is desirable to have greater stability and higher emission intensity at highest water content (fw = 99 vol%). At this point, we were intrigued to monitor the influence of carbon dots (CDs) as exogenous nanomaterial for the modification of physicochemical and AIE property of NDI-i FONPs. We have chosen CDs considering its similar dimension to that of organic nanoparticles as well as due to its intrinsic luminescence character. A reported protocol was followed to synthesize water soluble CDs (CD-i-iii, Figure 1a), where citric acid was used as carbon core and Na-salt of glycine (CD-i), 6-aminocaproic acid (CD-ii) and 11aminoundecanoic acid (CD-iii) as surface functionalities.33 The hydrophobicity of the CDs was modified from CD-i to CD-iii by increasing the alkyl chain length. All these CDs were found to be intrinsically blue fluorescent in water and showed excitation dependent emission behaviour (Figure 4). Change in excitation wavelength (from 300 nm to 400 nm) resulted in the red shift of emission maxima from 422 nm to 484 nm, 426 nm to 502 nm and 422 nm to 465 nm for CD-i-iii, respectively (Figure 4a-c). TEM images showed the dimension of CDs in the range of 6-8 nm (Figure 1c-e). In addition the DLS study of the surface functionalized CDs also revealed that the dimension of these CDs were in the range of 5-10 nm (Figure S4ac, Supporting Information). After addition of aqueous solutions of surface functionalized CDs 16 ACS Paragon Plus Environment

Page 16 of 48

Page 17 of 48 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

Langmuir

(0.5 g/mL) to the THF solution of NDI-i (20 M) (maintaining the solvent ratio 1:99 v/v, THF-water), the transparent aqueous solution turned into translucent (Figure S5, Supporting Information) similar to that of native NDI-i FONPs solution. Upon gradual increase in the concentration of the CD-i (up to 5 g/mL), no significant change in physical appearance of FONP solution was observed (Figure S5, Supporting Information). The hydrophilic character of CD-i and mismatch in the alkyl chain of CD-i (C-2) with NDI-i FONPs (C-10) possibly did not facilitate the hydrophobic interaction between these two nanoparticles. Hence, increase in the concentration of CD-i within in FONP solution could not enhance the translucency as well as stability of resulting conjugate. Notably, increasing concentration CD-ii and CD-iii with alkyl chain length of C-6 and C-11, respectively steadily improved the translucency of the FONP solution (Figure S5, Supporting Information). At higher concentration (1-5 g/mL) of CD-ii and CD-iii, the NDI-i FONP solution showed more translucency than that of the native FONP solution. Increase in the alkyl chain length (C-6 and C-11) on the CD surface might have facilitated the inter-chain hydrophobic interaction between CD (CD-ii/iii) and NDI-i FONPs to form a stable CD doped FONP conjugate. Higher is the chain length on the CD surface, better is the translucency and stability of the resulting nanoconjugate solution of CD-NDI-i-FONP. This observation indicates the functionalization (by varying alkyl chain length) on the CD surface has important role towards the formation of stable CD doped FONP solution. Effect of Surface Functionalized CDs on Size Tunability of NDI-i FONPs. Notable changes in the physical appearance of CD-doped FONP solutions prompted us to investigate the microstructural changes (if any) of organic nanoparticles upon incubation of surface functionalized CDs. In preceding section, the TEM images showed formation of NDI-i organic nanoparticles in fw = 99 vol% of diameter 30-40 nm (Figure 1b and S1a, Supporting Information). Doping of NDI-i organic nanoparticles (20 M) with 0.5 g/mL of CD-i (C-2 17 ACS Paragon Plus Environment

Langmuir 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

alkyl chain) did not yield any notable change in dimension as well as morphology of FONPs (Figure 5a). The size of the organic nanoparticles remained unaltered even with further increase in the concentration of CD-i (up to 5 g/mL) (Figure 5b,c). Probably, the mismatch in the alkyl chain length of CD-i and NDI-i could not facilitate inter-chain interaction between them and subsequently the dimension of the organic nanoparticles was found to be unaltered. Notable microstructural changes were observed for the organic nanoparticles upon doping with CDs having higher alkyl chain length on its surface. At 0.5 g/mL of CD-ii (C-6 alkyl chain), the size of the organic nanoparticles slightly increased in the range of 60-70 nm (Figure 5d). The dimension was of FONPs enhanced with gradual increase in the concentration of CD-ii and reached up to 100-190 nm, at [NDI-i] = 20 M and [CD-ii] = 5 g/mL (Figure 5e,f). Similarly, in case of CD-iii (C-11 alkyl chain), the modulation of dimension (100-125 nm) of organic nanoparticle as well as morphology was more prominent even at lowest concentration (0.5 g/mL) (Figure 5g). At the highest concentration of CD-iii, the diameter of the organic nanoparticles enhanced in the range of 250-300 nm and subsequently more fused structure was observed (Figure 5h,i). This change in dimension and morphology delineates that increasing the alkyl chain length on the CD surface (C-6 and C11) might have assisted the inter-chain hydrophobic interaction between CD and organic nanoparticles. As a result of this interaction, fused organic nanoparticles were formed at the higher concentration of CD-ii/iii. Enhancement of the hydrophobicity as well as concentration of the CDs aided more number of organic nanoparticle coming closer to form larger and fused spherical aggregates (Figure 5e,f,h,i). Hence, incubation of long hydrophobic alkyl chain containing CDs (both C-6 and C-11 alkyl chain) in FONP solution showed remarkable change in dimension and morphology (Scheme 1). The modification of dimension of organic nanoparticles in presence of surface functionalized CDs was also monitored by DLS study (Figure 6 and S6, Supporting 18 ACS Paragon Plus Environment

Page 18 of 48

Page 19 of 48 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

Langmuir

Information). The hydrodynamic diameter of NDI-i-CD-i organic nanoparticles (Dh = 30-50 nm at [NDI-i] = 20 M and [CD-i] = 5 g/mL) remained almost similar to the native NDI-i organic nanoparticles (Dh = 25-45 nm at [NDI-i] = 20 M) at fw = 99 vol% (Figure 6a and S1b, S6c Supporting Information). This observation supports the result found in TEM images (Figure 5a-c). Similarly, the DLS study also revealed that with gradual increase in the CDii/iii concentration, the Dh of the corresponding CD-doped organic nanoparticles accordingly increased (Figure 6b,c and S6d-i). At the highest concentration of both CD-ii and CD-iii, the Dh was found around 100-220 nm (Figure 6b and S6f, Supporting Information) and 150-295 nm, (Figure 6c and S6i, Supporting Information), respectively which is also in well concurrence with the respective TEM images (Figure 5d-i). This microscopic and DLS study confirmed that surface functionalized CDs have strong influence toward tuning the dimension of the NDI-i organic nanoparticles possibly through inter-chain hydrophobic interaction. Furthermore, we also monitored the influence of pH on the aggregation of organic nanoparticles in presence of CDs (only C-2 containing CD) by TEM and DLS study (Figure S7a,b, Supporting Information). At acidic pH (pH = 2.0, 20 mM KCl-HCl buffer) the carboxylate group (–COO-) is transformed to carboxylic acid group (–COOH). However, no significant change in dimension as well as morphology of organic nanoparticles was observed in presence of CDs at acidic pH (Figure S7a, Supporting Information). Similarly, the mean hydrodynamic diameter also remained similar (in the range of 20-40 nm) as found in case of native NDI-i organic nanoparticles (Figure S7b, Supporting Information). Presumably, at acidic pH, the –COOH functionalized could not effectively interact with the organic nanoparticles due to poor water solubility of such hydrophobic CDs (–COOH containing). Hence, no microstructural as well as dimensional change of CD doped organic nanoparticles was observed. This findings also suggested that the hydrophilic anionic CDs with varying


Langmuir 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

alkyl chain length (from C-2 to C-11) had major influence on the size tunability than –COOH functionalization at acidic pH. At this point we were curious to investigate how the organic particles are getting fused by hydrophobically surface functionalized CDs towards the formation of larger aggregates. For this purpose, instead of negative staining of the samples, the bright-field micrographs were taken under field-emission gun (FEG) beam at 200 kV and with higher divergence angle in order to restrict/minimize the potential damage of the material (Figure 7a-d). Also, the real-time reverse phase contrast technique micrographs were taken to have a better visualizing of the CDs in the self-assembled system (Figure 7e-h). In case of CD-i, no partial distribution of the carbon nanoparticles was observed around the surface of organic nanoparticles and the dimension of the organic nanoparticles remained unaltered (30-50 nm) (Figure 7a, e). In contrast, maximum distribution of CDs (CD-ii and CD-iii) was found at periphery of NDI-i organic nanoparticles and simultaneously the dimension of the spherical aggregates got enlarged (in the range of 200-250 nm, Figure 7b,c,f,g). This observation signifies that alkyl chain length on the CD surface plays a crucial role in the reorganization of the molecular packing of the aggregates. CD-ii (C-6 alkyl chain) and CD-iii (C-11 alkyl chain) surrounded the periphery of the organic nanoparticles possibly through inter-chain hydrophobic interaction between C-10 chain of NDI-i and long alkyl chain containing CDs. This favourable hydrophobic interaction facilitates the fusion of organic nanoparticles and consequently the augmentation in the dimension of organic nanoparticles. However, CD-i was unable to show any notable influence over the microstructure of NDI-i organic nanoparticles for mismatched hydrophobic interaction. Moreover, presence of large alkyl chain (especially C-11, CD-iii) on CD surface could efficiently anchor with the hydrophobically tailored (C-10 containing) NDI-i FONPs and bring more numbers of organic particles at close proximity to facilitate droplet-droplet interactions (Scheme 1). Hence, 20 ACS Paragon Plus Environment

Page 20 of 48

Page 21 of 48 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

Langmuir

organic nanoparticles got fused in presence of CD-iii where maximum distribution was observed at the junction of fused organic nanoparticles (Figure 7g, h). The influence of CD-iii on the fusion of organic particles observed even at 0.5 g/mL of the carbon dot due to the favourable inter-chain hydrophobic interaction. To further investigate the hydrophobic interactions upon doping of surface functionalized CDs in of NDI-i organic nanoparticles, we performed fluorescence spectroscopy study using 8-anilino-1-naphthalenesulfonic acid (ANS), a hydrophobic fluorescent probe. ANS showed weak emission (em) at 509 nm in aqueous medium upon excitation at 365 nm (Figure S8a-c, Supporting Information). In general, addition of ANS in NDI-i organic nanoparticle resulted in the enhancement in the emission intensity of ANS due to the hydrophobic interaction within the self-assembly of NDI-i. In case of NDI-i-CD-i system, inclusion of CD-i (1-5 g/mL) did not result any further change in emission intensity (Figure S8a, Supporting Information). On the other hand, the emission intensity of ANS was found to be notably enhanced with increasing concentration (1-5 g/mL) for both CD-ii and CD-iii included NDI-i organic nanoparticles (Figure S8b,c, Supporting Information). This enhancement in fluorescence intensity depicts that immediate vicinity of ANS are more hydrophobic in nature for CD doped self-assembled system (except NDI-i-CD-i) than native NDI-i organic nanoparticles. In case of NDI-i-CD-iii, highest emission intensity of ANS was found due to the maximum hydrophobicity in its environment owing to the inter-chain hydrophobic interaction. All these evidences support that surface functionalized CDs have notable influence on the size modulation as well as morphological change of NDI-i FONPs through hydrophobic interaction (Scheme 1). Effect of Surface Modified Carbon Dots on the AIE of Size Tunable NDI-i FONPs. Successful integration of surface modified CDs within self-assembly of organic nanoparticles 21 ACS Paragon Plus Environment

Langmuir 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

prompted us to investigate the emission behaviour of the NDI-i FONPs. In the present study, NDI-i FONPs showed comparatively poor AIE at fw = 99 vol% in comparison to that of at fw = 70 vol% (Figure 2b, 3a). However considering the potential biomedicinal applications of NDI-i FONPs, we carried out our investigation using NDI-i FONPs prepared at fw = 99 vol%. In presence of CD-i, the intensity of orange emission gradually decreased in comparison to the native NDI-i FONP solution (Figure 8). With gradual increase of the concentration CD-i (up to 5 g/mL), the orange emission got completely diminished and feeble blue emission was found upon UV-irradiation (Figure 8). On the other hand, upon doping of CD-ii/iii, the emission intensity of nanohybrid solution significantly increased compared to that of NDI-i FONP solution. This emission intensity steadily enhanced with increasing concentration of CD-ii and CD-iii (Figure 8). Among these CDs, CD-iii doped NDI-i FONP solution showed the strongest orange emission at the experimental highest concentration (5 g/mL). This notable change in the emission intensity indicates that the increase in the alkyl chain length (from C-2 to C-11) on the CD surface has strong influence on the AIE enhancement of the NDI-i FONPs at fw = 99 vol%. This change in photophysical property of NDI-i FONPs upon addition of surface functionalized CDs was further envisaged by investigating the photoluminescence spectra. In preceding observation it was found that NDI-i showed AIE via excimer formation having em = 580 nm (orange emission) (Figure 3a). In presence of surface functionalized CDs of varying concentration (10 ng/mL to 5 g/mL) to the FONP solution (at fw = 99 vol%), the excimer band intensity was judiciously tuned (Figure 9a-c). Upon addition of CD-i, the excimer band intensity at 580 nm was initially reduced at the lowest concentration of CD-i (10 ng/mL) (Figure 9a). With gradual increase in concentration of CD-i (up to 0.2 g/mL), the intensity of excimer band steadily decreased (Figure 9a). The existence of excimer of NDI-i in presence of CD-i was observed up to [CD-i] = 1 g/mL without any significant 22 ACS Paragon Plus Environment

Page 22 of 48

Page 23 of 48 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

Langmuir

amplification of excimer emission intensity (Figure 9a). Beyond 1 g/mL of CD-i, the excimer band for AIE got completely diminished presumably due to the fluorescence quenching property of CD-i and characteristic emission band of CD-i was observed at 425 nm (ex = 350 nm). CD-i could not interact with NDI-i molecules due to mismatch in the inter-chain hydrophobic interaction between CD-i (C-2 alkyl) and NDI-i (C-10 alkyl) molecules. Hence, this short chain CDs (CD-i) were unable to accumulate more number of hydrophobically tailored NDI-i molecules around their surface to show enhanced AIE. Thus, the quenching effect of CD-i got more predominant over the enhancement of AIE. This observation supports the change in photoluminescence behaviour of CD-i doped NDI-i FONPs as shown in Figure 8. Moreover, the quenching of orange emission of NDI-i FONPs was also confirmed from the photoluminescence spectra due to the reduction of excimer band in presence of CD-i (Figure 9a). On the other hand, when the alkyl chain length on the CD surface was increased to C-6 and C-11, the excimer band intensity of NDI-i FONPs enhanced even at very low concentration (10 ng/mL) of CD-ii and CD-iii (Figure 9b,c). At the lowest concentration (10 ng/mL) of CDs, The enhancement in the excimer band intensity was more prominent in case of CD-iii doped FONPs than CD-ii doped system (Figure 9b,c). The emission intensity of the excimer band steadily enhanced with increasing concentration of both CD-ii/CD-iii (Figure 9b, c). At 5 g/mL (experimentally used highest concentration of CD), NDI-i FONPs showed ~3.6-fold (CD-ii doped) and ~5-fold (CD-iii doped) enhancement in the fluorescence intensity than that of native NDI-i (Figure 9b-d). The observed highest excimer band intensity for CD-iii doped NDI-i FONPs nanoconjugate was also in accordance to the photographic images FONP solution upon UV-irradiation (Figure 8 and Figure 9c,d). Increase in the alkyl chain length on CD surface as well as concentration of CDs might have facilitated the accumulation of more NDI-i around the CD surface via favourable inter-chain hydrophobic interaction toward the formation of fused aggregates (Scheme 1). Consequently, 23 ACS Paragon Plus Environment

Langmuir 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

the extent of intramolecular charge transfer (ICT) between the -donor (naphthyl group) and -acceptor (NDI core) residue became more efficient to exhibit amplified AIE. In addition the effect of surface functionalized CDs on the enhancement of emission intensity was also monitored in presence of a well-known fluorescence quencher e.g., methyl viologen dichloride hydrate (MV) (Figure S9, Supporting Information). In both the case (for NDI-i FONPs, [NDI-i] = 20 M and NDI-i-CD-iii FONPs, [NDI-i] = 20 M, [CD-iii] = 0.5 g/mL), the excimer band intensity at em = 580 nm got gradually quenched with increasing the concentration of MV (from 5 M to 20 M) (Figure S9a,b, Supporting Information). However, the extent of quenching of excimer band intensity is lesser for NDI-i-CD-iii FONPs than native NDI-i FONPs at the highest concentration (20 M) of MV due to enhanced AIE of CD-iii doped NDI-i FONPs. This observation suggests that the long alkyl chain containing CDs (especially CD-iii) has the strong influence on the enhancement of AIE even in presence of a fluorescence quencher molecule (Inset of Figure S9b, Supporting Information). This photophysical change was also reflected in the corresponding quantum yield (QY) of the CD doped FONPs (Figure 9e). QY of NDI-i FONPs was estimated to be 1.98% (with quinine sulfate hydrate as a standard). In presence of CD-i, the QY value reduced to 1.03%. Interestingly, the QY value enhanced to 4.07% and 5.32% upon doping with CD-ii and CD-iii, respectively. This result corroborated the effect of surface functionalized CDs on the emission behaviour of NDI-i FONPs. The change in emission intensity of CD doped NDI-i FONPs was further correlated with time-resolved study (Figure 9f). In presence of CDi, the average life-time () of NDI-i FONPs (ex = 375 nm, mon = 580 nm) was found to be 0.33 ns which is much less than the average life-time value of native NDI-i FONPs ( = 0.81 ns) (Table 1). However, was estimated 1.01 ns and 1.15 ns for NDI-i-CD-ii and NDI-i-CD-iii FONPs, respectively (Table 1). The fluorescence life-time CD-i doped NDI-i 24 ACS Paragon Plus Environment

Page 24 of 48

Page 25 of 48 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

Langmuir

FONPs was shortened than native NDI-i FONPs due to quenching of excimer band intensity by CD-i (Figure 9a). On the other hand, in presence of CD-ii and CD-iii the excimer band intensity (em = 580 nm) of NDI-i notably enhanced and subsequently the life-time value also increased than the native NDI-i FONPs. This overall spectroscopic observation clearly suggests that surface functionalized CDs have strong influence in regulating the AIE process of NDI-i FONPs by the formation of fused self-aggregates of organic nanoparticle via hydrophobic interaction. Effects of Surface Functionalized CDs on Stability of NDI-i FONPs. Next we were intrigued to investigate the stability of the soft-nanohybrid (except CD-i doped system) upon the formation of fused larger aggregates owing to the inclusion of surface modified CDs (CDii/iii). To this end, we recorded time dependent UV-visible and photoluminescence spectra of the nanocomposites at fw = 99 vol% (Figure S10a,b, Supporting Information). The relative absorbance (A/A0 at max = 384 nm) as well as relative emission intensity (I/I0 at em = 580 nm) was monitored at different time scale (up to 7 days) in absence and presence of CD of varying concentration (Figure S10a, Supporting Information). In case of native NDI-i FONPs (20 M), the absorbance drastically reduced by 60% only within 24 h (Figure S10a, Supporting Information). In contrast, for CD doped NDI-i FONPs the absorbance remained same and marginally reduced by 10% for CD-iii and CD-ii doped system, respectively. At 5 g/mL of doped CDs, the absorbance maximally reduced by 50 and 30% after 7 days, which indicates that the inclusion of surface modified CDs within self-assembled organic particles improved the overall stability of the nanohybrid FONPs. The superior stability was noted when the NDI-i FONPs was doped with CD-iii with increasing concentration (Figure S10a, Supporting Information). We also measured the relative fluorescence intensity (I/I0) of NDI-i FONPs with the varying concentration (0 to 5 g/mL) of added CDs and the similar trend in the stability of FONP-CD conjugates was observed (Figure S10b, Supporting Information). 25 ACS Paragon Plus Environment

Langmuir 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

Hydrophobically surface modified CDs (CD-ii and CD-iii) play an important role to develop highly water dispersible stable FONP-CD conjugates. Cytocompatibility and Long Term Bioimaging of NDI-i-CD-iii FONPs. Next, we intended to make use of these highly emissive CD doped AIE-gens for cellular tracking. NDI-i-CD-iii was chosen for bioimaging probe for its highest emission intensity as well as superior stability. We have checked the cytocompatibility of both NDI-i and NDI-i-CD-iii FONPs (1:99, v/v THF-water) in B16F10 mammalian cells (Figure S11a,b, Supporting Information). For native NDI-i FONPs the experimental concentration range was of 5-40 M while in case of NDI-i-CD-iii the concentration of CD-iii was varied from 1-5 g/mL keeping the NDI-i FONPs concentration fixed at 20 M. Both the NDI-i and NDI-i-CD-iii showed 85-94 % cell viability after 24 h incubation at their experimental concentration ([NDI-i] = 20 M for NDI-i FONPs and [NDI-i] = 20 M, [CD-iii] = 5 g/mL for NDI-i-CDiii FONPs). The ability of the intrinsically fluorescent, aggregated NDI-i and NDI-i-CD-iii FONPs to label the mammalian cells was investigated by fluorescence microscopy and flow cytometric analysis (Figure 10, S12, Supporting Information). B16F10 cells were incubated with both NDI-i (20 M) and NDI-i-CD-iii ([NDI-i] = 20 M and [CD-iii] = 5 g/mL) for the time period of 6-24 h. The fluorescence microscopic images in both cases showed red fluorescence inside the B16F10 cells after 6 h incubation (Figure 10e,m). The emission intensity of the NDI-i-CD-iii incubated B16F10 cells is notably higher (having mean fluorescence intensity of 4751) than that of the native NDI-i FONP doped cells (having mean fluorescence intensity of 2996) (Figure 10e,m and Figure S12, Supporting Information). Also more number of cells were found to be labelled by NDI-i-CD-iii FONPs. Interestingly, emission intensity of NDI-i-CD-iii was remaining mostly unaltered over the time period of 24 26 ACS Paragon Plus Environment

Page 26 of 48

Page 27 of 48 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

Langmuir

h (mean fluorescence intensity of 4059) (Figure 10n-p and Figure S12, Supporting Information). However, the red emission intensity for native NDI-i doped in B16F10 cells steadily depleted beyond 6 h and almost no fluorescence signal was observed after 24 h (mean fluorescence intensity of 495) (Figure 10f-h and Figure S12, Supporting Information). These observations depicted that NDI-i-CD-iii nanohybrid is much efficient bioimaging probe and could retain their stability in the cells over long period of time than that of the native NDI-i FONPs. CONCLUSIONS In conclusion, we have designed L- tyrosine tagged hydrophobically tailored NDI derivative (NDI-i) which formed organic nanoparticles in THF-water binary solvent mixture. NDI-i showed aggregation induced orange emission via excimer formation in combination with ICT at 580 nm upon excitation at 350 nm. At higher water percentage (beyond fw = 70 vol%), the emission intensity gradually reduced because of poor water dispersibility of NDI-i FONPs. This poor emission intensity of the NDI-i FONPs at fw = 99 vol%, got remarkably enhanced upon addition of surface functionalized CDs of varying alkyl chain length (except CD-i). Increasing the alkyl chain length on CD surface facilitated the inter-chain hydrophobic interaction between the organic nanoparticles and surface functionalized CDs to form larger size fused CD-doped FONPs. This inter-droplet interaction in presence of long chain containing (C-6 and C-11) CDs assisted maximum accumulation of NDI-i around CD surface. Hence, the extent of ICT between -donor and -acceptor residue became more efficient to exhibit amplified AIE of the FONP-CD nano-conjugates. In presence of C-11 alkyl chain containing CD, the FONP solution showed the brightest orange emission with superior stability. This water dispersible, orange emitting, cytocompatible NDI-i-CD-iii FONPs were explored for the long-term cellular imaging.


Langmuir 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

Supporting Information Available: Synthetic scheme and characterization data of NDI-i, TEM image and DLS of NDI-i FONPs in (1:99, v/v) THF-water, UV-visible spectra of NDIi, solvent dependent 1H-NMR spectra of NDI-i, excitation dependent emission spectra of CDi-iii, DLS study of CDs, photographs of change in physical appearance of NDI-i upon CD incubation, DLS histogram plots of CD doped NDI-i organic nanoparticles, TEM image and DLS plot of CD-i doped NDI-i organic nanoparticles at pH = 2.0, fluorescence spectra of NDI-i FONPs and NDI-i-CD-iii FONPs in presence of methyl viologen dichloride hydrate, fluorescence spectra of ANS in presence of NDI-i FONPs and CD doped NDI-i FONPs, temporal evolution of the absorbance and fluorescence intensity NDI-i FONPs in presence of CD-ii/iii, MTT assay of NDI-i and NDI-i-CD-iii FONPs in B16F10 cells, corresponding flow cytometric histogram plots. ACKNOWLEDGEMENTS P.K.D. is thankful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), India, for financial assistance (No. EMR/2017/000656). P.C. acknowledges CSIR, India for Research Fellowship. We are thankful to Saheli Sarkar for helpful discussion. REFERENCES (1) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang. B. Z. Aggregation-induced emission of 1-methyl-1,2,3,4,5pentaphenylsilole. Chem. Commun. 2001, 1740-1741. (2) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced emission. Chem. Soc. Rev. 2011, 40, 5361-5388. (3) Kumar, M.; George, S. J. Green fluorescent organic nanoparticles by self-assembly induced enhanced emission of a naphthalene diimide bolaamphiphile. Nanoscale 2011, 3, 2130-2133. 28 ACS Paragon Plus Environment

Page 28 of 48

Page 29 of 48 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

Langmuir

(4) Kaeser, A.; Fischer, I.; Abbel, R.; Besenius, P.; Dasgupta, D.; Gillisen, M. A. J.; Portale, G.; Stevens, A. L.; Herz, L. M.; Schenning, A. P. H. J. Side chains control dynamics and selfsorting in fluorescent organic nanoparticles. ACS Nano 2013, 7, 408-416. (5) Kaeser, A.; Schenning, A. P. H. J. Fluorescent nanoparticles based on self-assembled πconjugated systems. Adv. Mater. 2010, 22, 2985-2997. (6) Choudhury, P.; Das, K.; Das, P. K. L-phenylalanine-tethered, naphthalene diimide-based, aggregation-induced, green-emitting organic nanoparticles. Langmuir 2017, 33, 4500-4510. (7) Chen, H. Y.; Lam, W. Y.; Luo, J. D.; Ho, Y. L.; Tang, B. Z.; Zhu, D. B.; Wong, M.; Kwok, H. S. Highly efficient organic light-emitting diodes with a silole-based compound. Appl. Phys. Lett. 2002, 81, 574-576. (8) Wang, F.; Han, M. Y.; Mya, K. Y.; Wang, Y.; La, Y. H. Aggregation-driven growth of size-tunable organic nanoparticles using electronically altered conjugated polymers. J. Am. Chem. Soc. 2005, 127, 10350-10355. (9) Cui, Q.; Yang, Y.; Yao, C.; Liu, R.; Li, L. Aggregation-induced energy transfer of conjugated polymer materials for ATP sensing. ACS Appl. Mater. Interfaces 2016, 8, 3557835586. (10) Zhang, C.; Jin, S.; Li, S.; Xue, X.; Liu, J.; Huang, Y.; Jiang, Y.; Chen, W. Q.; Zou, G.; Liang, X. J. Imaging intracellular anticancer drug delivery by self-assembly micelles with aggregation-induced emission (AIE micelles). ACS Appl. Mater. Interfaces 2014, 6, 52125220. (11) Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible nanoparticles with aggregation-induced emission characteristics as far-red/near-infrared fluorescent bioprobes for in vitro and in vivo imaging applications. Adv. Funct. Mater. 2012, 22, 771-779.


Langmuir 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

(12) Li, K.; Liu, B. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem. Soc. Rev. 2014, 43, 6570-6597. (13) Ng, K. K.; Zheng, G. Molecular interactions in organic nanoparticles for phototheranostic applications. Chem. Rev. 2015, 115, 11012-11042. (14) Nakanishi, H.; Oikawa, H. Reprecipitation method for organic nanocrystals. In Single Organic Nanoparticles; Masuhara, H., Nakanishi, H., Sasaki, K., Eds.; Springer-Verlag: Berlin, 2003; pp 17-31. (15) Zhang, X.; Zhang, X.; Wang, S.; Liu, M.; Tao, L.; Wei, Y. Surfactant modification of aggregation-induced emission material as biocompatible nanoparticles: facile preparation and cell imaging. Nanoscale 2013, 5, 147-150. (16) Zhang, X.; Wang, K.; Liu, M.; Zhang, X.; Tao, L.; Chen, Y.; Wei, Y. Polymeric AIEbased nanoprobes for biomedical applications: recent advances and perspectives. Nanoscale 2015, 7, 11486-11508. (17) Wang, D.; Qian, J.; He, S.; Park, J. S.; Lee, K. S.; Han, S.; Mu, Y. Aggregationenhanced fluorescence in PEGylated phospholipid nanomicelles for in vivo imaging. Biomaterials 2011, 32, 5880-5888. (18) Zhu, Z. F.; Zhao, X. Y.; Qin, W.; Chen, G. D.; Qian, J.; Xu, Z. P. Fluorescent AIE Dots encapsulated organically modified silica (ORMOSIL) nanoparticles for two-photon cellular imaging. Sci. China: Chem. 2013, 56, 1247-1252. (19) Grabowski, N.; Hillaireau, H.; Vergnaud, J.; Tsapis, N.; Pallardy, M.; Kerdine-Römer, S.; Fattal, E. Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages. Int. J. Pharm. 2015, 482, 75-83. (20) Hamilton Jr., R. F.; Thakur, S. A.; Holian, A. Silica binding and toxicity in alveolar macrophages. Free Radic. Biol. Med. 2008, 44, 1246-1258.


Page 30 of 48

Page 31 of 48 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

Langmuir

(21) Bhattacharya, S.; Samanta, S. K. Soft-nanocomposites of nanoparticles and nanocarbons with supramolecular and polymer gels and their applications. Chem. Rev. 2016, 116, 1196712028. (22) Choudhury, P.; Mandal, D.; Brahmachari, S.; Das, P. K. Hydrophobic end-modulated amino-acid-based neutral hydrogelators: structure-specific inclusion of carbon nanomaterials. Chem. Eur. J. 2016, 22, 5160-5172. (23) Dinda, S.; Mandal, D.; Sarkar, S.; Das, P. K. Self-assembled vesicle-carbon nanotube conjugate formation through a boronate-diol covalent linkage. Chem. Eur. J. 2017, 23, 15194-15202. (24) Jiao, T.; Zhao, H.; Zhou, J.; Zhang, Q.; Luo, X.; Hu, J.; Peng, Q.; Yan, X. Self-assembly reduced graphene oxide nanosheet hydrogel fabrication by anchorage of chitosan/silver and its potential efficient application toward dye degradation for wastewater treatments. ACS Sustainable Chem. Eng. 2015, 3, 3130-3139. (25) Fan, Z.; Liu, B.; Wang, J.; Zhang, S.; Lin, Q.; Gong, P.; Ma, L.; Yang, S. A novel wound dressing based on Ag/graphene polymer hydrogel: effectively kill bacteria and accelerate wound healing. Adv. Funct. Mater. 2014, 24, 3933-3943. (26) Hu, Z.; Pantoş, G. D.; Kuganathan, N.; Arrowsmith, R. L.; Jacobs, R. M. J.; KociokKöhn, G.; O’Byrne, J.; Jurkschat, K.; Burgos, P.; Tyrrell, R. M.; Botchway, S. W.; Sanders, J. K. M.; Pascu, S. I. Interactions between amino acid-tagged naphthalenediimide and single walled carbon nanotubes for the design and construction of new bioimaging probes. Adv. Funct. Mater. 2012, 22, 503-518. (27) Pantoş, G. D.; Wietor, J. L.; Sanders, J. K. M. Filling helical nanotubes with C60. Angew. Chem. Int. Ed. 2007, 46, 2238-2240. (28) Zhu, Z.; Qian, J.; Zhao, X.; Qin, W.; Hu, R.; Zhang, H.; Li, Dongyu; Xu, Z.; Tang, B. Z.; He, S. Stable and size-tunable aggregation-induced emission nanoparticles encapsulated 31 ACS Paragon Plus Environment

Langmuir 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

with nanographene oxide and applications in three-photon fluorescence bioimaging. ACS Nano 2016, 10, 588-597. (29) Hu, M.; Yang, Y.; Gu, X.; Hu, Y.; Du, Z.; Wang, C. Novel nanocomposite hydrogels consisting of C‐dots with excellent mechanical properties. Macromol. Mater. Eng. 2015, 300, 1043-1048. (30) Konwar, A.; Gogoi, N.; Majumdar, G.; Chowdhury, D. Green chitosan-carbon dots nanocomposite hydrogel film with superior properties. Carbohydr. Polym. 2015, 115, 238245. (31) Sarkar, S.; Dinda, S.; Choudhury, P.; Das, P. K. Self-assembly of surface functionalized amphiphilic carbon dots: tuning in morphological manifestations. Soft Matter 2019, 15, 28632875. (32) Wang, Y.; Hu, A. Carbon quantum dots: synthesis, properties and applications. J. Mater. Chem. C 2014, 2, 6921-6939. (33) Chen, X.; Jin, Q.; Wu, L.; Tung, C.; Tang, X. Synthesis and unique photoluminescence properties of nitrogen-rich quantum dots and their applications. Angew. Chem. Int. Ed. 2014, 53, 12542-12547. (34) Sarkar, S.; Das, K.; Das, P. K. Hydrophobically tailored carbon dots toward modulating microstructure of reverse micelle and amplification of lipase catalytic response. Langmuir 2016, 32, 3890-3900. (35) König, K. Multiphoton microscopy in life sciences. J. Microsc. 2000, 200, 83-104. (36) Choudhury, P.; Sarkar, S.; Das, P. K. Tunable aggregation-induced multicolor emission of organic nanoparticles by varying the substituent in naphthalene diimide. Langmuir 2018, 34, 14328-14341.


Page 32 of 48

Page 33 of 48 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

Langmuir

(37) Kobaisi, M. A.; Bhosale, S. V.; Latham, K.; Raynor, A. M.; Bhosale, S. V. Functional naphthalene diimides: synthesis, properties, and applications. Chem. Rev. 2016, 116, 1168511796. (38) Bhosale, S. V.; Jani, C. H.; Langford, S. J. Chemistry of naphthalene diimides. Chem. Soc. Rev. 2008, 37, 331-342. (39) Sakai, N.; Mareda, J.; Vauthey, E.; Matile, S. Core-substituted naphthalenediimides. Chem. Commun. 2010, 46, 4225-4237. (40) He, T.; Stolte, M.; Würthner, F. Air-stable n-channel organic single crystal field-effect transistors based on microribbons of core-chlorinated naphthalene diimide. Adv. Mater. 2013, 25, 6951-6955. (41) Molla, M. R.; Ghosh, S. Hydrogen-bonding-mediated J-aggregation and white-light emission from a remarkably simple, single-component, naphthalenediimide chromophore. Chem. Eur. J. 2012, 18, 1290-1294. (42) Zong, L.; Xie, Y.; Li, Q.; Li, Z. A new red fluorescent probe for Hg2+ based on naphthalene diimide and its application in living cells, reversibility on strip papers. Sens. Actuator B Chem. 2017, 238, 735-743. (43) Nalluri, S. K. M.; Berdugo, C.; Javid, N.; Frederix, P. W. J. M.; Ulijn, R. V. Biocatalytic self-assembly

of

supramolecular

charge-transfer

nanostructures

based

on

n-type

semiconductor-appended peptides. Angew. Chem. Int. Ed. 2014, 53, 5882-5887. (44) Kumar, M.; George, S. J. Spectroscopic probing of the dynamic self-assembly of an amphiphilic naphthalene diimide exhibiting reversible vapochromism. Chem. Eur. J. 2011, 17, 11102-11106. (45) Bell, T. D. M.; Bhosale, S. V.; Forsyth, C. M.; Hayne, D.; Ghiggino, K. P.; Hutchison, J. A.; Jani, C. H.; Langford, S. J.; Lee, M. A. P.; Woodward, C. P. Melt-induced fluorescent signature in a simple naphthalenediimide. Chem. Commun. 2010, 46, 4881-4883. 33 ACS Paragon Plus Environment

Langmuir 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

(46) Shao, H.; Nguyen, T.; Romano, N. C.; Modarelli, D. A.; Parquette, J. R. Self-assembly of 1-D n-type nanostructures based on naphthalene diimide-appended dipeptides. J. Am. Chem. Soc. 2009, 131, 16374-16376. (47) Lippert, E. Dipolmoment und elektronenstruktur von angeregten molekülen. Z. Naturforsch., A: Phys. Sci. 1955, 10, 541-545. (48) Mataga, N.; Kaifu, Y.; Koizumi, M. Solvent effects upon fluorescence spectra and the dipolemoments of excited molecules. Bull. Chem. Soc. Jpn. 1956, 29, 465-470.


Page 34 of 48

Page 35 of 48 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

Langmuir

Table 1. Lifetime values of NDI-i and CD-doped NDI-i in THF-water binary solvent mixture (λex = 375 nm) Sample

NDI-i

fraction of water (fw)

monitored

1 (ns)

2 (ns)

3 (ns)

(nm)

(a1)

(a2)

(a3)

0 vol%

404

1.10

0.03

5.70

(0.008)

(0.991)

(0.001)

2.11

7.72

0.27

(0.105)

(0.046)

(0.849)

1.56

0.24

6.71

(0.032)

(0.961)

(0.007)

2.22

9.61

0.20

(0.071)

(0.071)

(0.858)

2.39

8.73

0.20

(0.087)

(0.087)

(0.826)

99 vol%

NDI-i-CD-i

NDI-i-CD-ii

NDI-i-CD-iii

99 vol%

99 vol%

99 vol%

580

580

580

580


Average lifetime ns 0.04

0.81

0.33

1.01

1.15

Langmuir 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 36 of 48

Table 2. Photophysical property of NDI-ia Solvent

Δf

λab (nm)

λem (nm)

Δν (cm−1)

Benzene

0.002

383

581

8898.0

Toluene

0.013

382

582

8995.9

o-Xylene

0.027

382

585

9084.0

Chloroform

0.150

382

606

9676.3

Dichloromethane

0.217

382

620

10049

aAbbreviation:

Δf = solvent polarity parameters, λab = absorption maximum, λem = emission maximum, Δν = Stokes shift. Excitation wavelength (ex = 350 nm)


Page 37 of 48 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

Langmuir

Scheme 1. Schematic presentation of enhancement of aggregation induced emission of size tunable NDI-i FONPs in presence of long alkyl chain (C-6 and C-11) containing CDs. AIE: aggregation induced emission, ICT: intramolecular charge transfer.


Langmuir 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

Figure 1. (a) Chemical structure of NDI-i and surface functionalized carbon dots (CD-i-iii). (b) TEM image (negatively stained) of NDI-i (20 M) in (1:99, v/v) THF-water. (c-e) TEM images of surface functionalized carbon dots (CD-i-iii), respectively.


Page 38 of 48

Page 39 of 48 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

Langmuir

Figure 2. (a) Photographs of change in physical appearance of the NDI-i (20 M) solutions with increasing water content in THF with a black background. (b) Photographs of change in emission color of NDI-i (20 M) with increasing water content in THF upon UV-light irradiation (ex = 365 nm).


Langmuir 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

Figure 3. (a) Fluorescence spectra of NDI-i (20 M) in THF-water solvent mixture of varying ratio (path length = 10 mm, ex = 350 nm). (b) A plot of the relative emission intensity (I/I0) vs fraction of water in THF of NDI-i; I0 = Emission intensity at 404 nm in pure THF solution. (c) TCSPC decay profile of NDI-i with 375 nm excitation in different solvent compositions of THF-water. (d) Absorption and (e) emission spectra of NDI-i (20 M) in different solvents of varying polarities. (f) Plot of Stokes shift (Δν) of NDI-i vs solvent polarity parameter (Δf).


Page 40 of 48

Page 41 of 48 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

Langmuir

Figure 4. Excitation dependent emission spectra of (a) CD-i, (b) CD-ii and (c) CD-iii.


Langmuir 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

Figure 5. (a-i) Negatively stained TEM images of NDI-i-CD-i, NDI-i-CD-ii and NDI-i-CDiii FONPs ([NDI-i] = 20 M) in (1:99, v/v) THF-water of varying amount of surface functionalized CDs. [CD-i]/[CD-ii]/CD-iii] = 0.5 g/mL (a, d, g), 1.0 g/mL (b, e, h), 5.0 g/mL (c, f, i), respectively.


Page 42 of 48

Page 43 of 48 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

Langmuir

Figure 6. (a-c) DLS study of NDI-i FONPs in absence and presence of surface functionalized carbon dots (CD-i/ii/iii) of varying amounts (0.5-5.0 g/mL).


Langmuir 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

Figure 7. (a-h) TEM images (without negative staining) of surface functionalized CD doped NDI-i FONPs. (a-c) bright field micrographs and (e-g) real-time reverse phase contrast micrographs of NDI-i FONPs doped with CD-i, CD-ii and CD-iii, respectively. (d, h) Magnified bright field and real-time reverse phase contrast micrographs of NDI-i FONPs doped with CD-iii. In each case, [NDI-i] = 20 M and [CD-i]/[CD-ii]/[CD-iii] = 5.0 g/mL.


Page 44 of 48

Page 45 of 48 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

Langmuir

Figure 8. Photographs of change in emission color and intensity of NDI-i (20 M) FONPs with increasing concentration of different surface functionalized CDs (0.5-5.0 g/mL) in (1:99, v/v) THF-water upon UV-light irradiation (ex = 365 nm).


Langmuir 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

Figure 9. (a-c) Photoluminescence spectra of NDI-i in (1:99, v/v) THF-water upon doping with varying amount of CD-i, CD-ii and CD-iii, respectively. (d) A plot of the relative emission intensity (I/I0) of NDI-i vs concentration of surface functionalized CDs in (1:99, v/v) THF-water; I0 = Emission intensity of NDI-i in fw = 99 vol%. (e) Quantum yield values of NDI-i in absence and presence of CDs (f) TCSPC decay profile of CD doped NDI-i with 375 nm excitation in (1:99, v/v) THF-water.


Page 46 of 48

Page 47 of 48 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

Langmuir

Figure 10. Bright field images of B16F10 cells upon incubation with NDI-i FONPs (a-d) and NDI-i-CD-iii FONPs (i-l) at different time interval (6 h, 12 h, 18 h and 24 h, respectively). Fluorescence microscopic images of B16F10 cells upon incubation with NDI-i FONPs (e-h) and NDI-i-CD-iii FONPs (m-p) at different time interval (6 h, 12 h, 18 h and 24 h, respectively). For NDI-i FONPs: [NDI-i] = 20 M; for NDI-i-CD-iii FONPs: [NDI-i] = 20 M and [CD-iii] = 5.0 g/mL. Scale bars correspond to 20 μm.


Langmuir 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

For Table of Contents Use Only

Carbon Dots Stimulated Amplification of Aggregation Induced Emission of Size Tunable Organic Nanoparticles Pritam Choudhury and Prasanta Kumar Das* School of Biological Sciences, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700032, India.


Page 48 of 48

Carbon Dots-Stimulated Amplification of Aggregation-Induced

Recommend Documents