Near-Infrared Fluorescent Nanoprobes for Revealing the Role of

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Near-Infrared Fluorescent Nanoprobes for Revealing the Role of Dopamine in Drug Addiction Peijian Feng, Yulei Chen, Lei Zhang, Cheng-Gen Qian, Xuanzhong Xiao, Xu Han, and Qun-Dong Shen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12005 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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ACS Applied Materials & Interfaces

Near-Infrared Fluorescent Nanoprobes for Revealing the Role of Dopamine in Drug Addiction Peijian Feng,1# Yulei Chen,1# Lei Zhang,2 Cheng-Gen Qian,1 Xuanzhong Xiao,1 Xu Han,1 and Qun-Dong Shen1,* 1. Key Laboratory of High Performance Polymer Materials and Technology of MOE, Collaborative Innovation Center of Chemistry for Life Sciences, Department of Polymer Science and Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, China. 2. Department of Biomedical Engineering, College of Engineering and Applied Science, Nanjing University, Nanjing 210093, China. Keywords: brain activity, drug addiction, dopamine-responsive, near-infrared fluorescence, functional neuroimaging Abstract: Brain imaging techniques enable visualizing the activity of central nervous system without invasive neurosurgery. Dopamine is an important neurotransmitter. Its fluctuation in brain leads to a wide range of diseases and disorders, like drug addiction, depression, and Parkinson disease. We designed near-infrared fluorescence dopamine-responsive nanoprobes (DRNs) for brain activity imaging during drug abuse and addiction process. Based on light-induced electron transfer between DRNs and dopamine and molecular wire effect of the DRNs, we can track

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dynamical change of the neurotransmitter level in the physiological environment and the releasing of the neurotransmitter in living dopaminergic neurons in response to nicotine stimulation. The functional near-infrared fluorescent (NIRF) imaging can dynamically track the dopamine level in the mice mid-brain under normal or drug-activated condition and evaluate the long-term effect of addictive substances to the brain. This strategy has the potential for studying neural activity in physiological condition. 1. Introduction Brain imaging techniques, e.g., magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT), permit visualizing activity of central nervous system without invasive neurosurgery. 1 A variety of neuroimaging modalities are also important in the early detection and surveillance of the neural disorders like Alzheimer’s and Parkinson’s diseases 2-3 and delineating brain tumor margins for accurate surgical resection 4. Addiction is a chronic and relapsing brain disease, where drugs or chemicals interfere with the way that neurons normally send, receive, and process information through the network.

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Mid-

brain dopaminergic nerve cells in ventral tegmental area (VTA) are crucial to reward processing. 6-7

The addictive substances or stimulants, such as nicotine, alcohol, marijuana, and cocaine, are

able to increase or disrupt the level of dopamine, the important massager in neural activity. Brain imaging technologies are actively involved in evaluating the importance of dopamine in drug reward/addiction. Using radiolabeled drug, PET images reveal local distribution of drug binding sites and the mechanism of dopamine involved in the reinforcing effects of stimulant drugs.

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Such functional imaging technique is sensitive to sub-nanomolar concentrations of radio-tracers, and has spatial resolution of millimeter. Functional magnetic resonance imaging (fMRI) makes


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use of contrast agents to detect dopamine signaling in ventral striatum, pathway in cocaine addiction

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and elucidate neural

. The fMRI-based molecular mapping provides a spatial

resolution of 100 µm and a detection limit of tens µM. Taking advantage of the redox activity of dopamine, the electrochemical techniques can sense the neurotransmitter in a direct but invasive manner.

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The excellent temporal resolution (sub-second timescale) with micron spatial

resolution and high sensitivity permit detection of dopamine fluctuation during cocaine selfadministration.14 Optical imaging is an essential alternative for brain imaging. However, at present, diffraction-limited resolution of optical imaging makes it difficult to image the whole brain in a single cell, and the penetration depth is limited due to light scattering. It also remains a challenge for imaging brain activity in freely behaving animals. The use of longer wavelength with less scattering is a common solution for deep-tissue imaging.

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For revealing the neural

activity, it is of great value to combine the virtues of dopamine-specified imaging and detecting techniques. Here we introduce near-infrared fluorescent (NIRF) and dopamine-responsive nanoprobes (designated DRNs) for in vitro and in vivo imaging of neural activity, especially for the tracking of dopamine level in stimulant-induced mice brain (Scheme 1). Optical imaging which uses near-infrared light offers the advantage of deep tissue penetration, less background autofluorescence, and low light scattering. Such non-radioactive technique has been widely used to improve cancer surgery outcomes, and enhance our understanding of biological activity18-21 or on-target effects of the drugs22-25. It can also generate two-dimensional images of cerebral hemodynamics and map distributed brain function and networks.26-27 To investigate dopamine signals in drug addiction, the DRNs are delivered into mice brain by standard intra-ventricular injections

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. We observe neural activity of the normal and addicted brain by near-infrared


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emission of the DRNs, which have phenylboronic acid (PBA) tags for adsorption of the neurotransmitter through specific recognition of boronic acid to dopamine molecule30-32. Due to the redox activity of dopamine, photo-induced electron transfer between the neurotransmitter and the DRNs will result in fluorescence quenching behavior, indicating the binding event instantly.33-34 To reduce immunogenic response and cytotoxicity, the surface of the nanoparticles is linked with short-chain-length polyethylene glycol (PEG) 35-40.

Scheme 1. The dopamine-responsive near-infrared fluorescent (NIRF) nanoprobes (DRNs) for in vitro and in vivo imaging of neural activity in mid-brain of the normal and addicted mice. 2. Experimental Section Materials and animals: All chemicals were purchased from Sigma-Aldrich unless otherwise specified. Compound 1 was prepared according to previous work.41 Compound 2 and 3 were afforded by Beijing Allmers Chemical. Polyethylene glycol methyl ether succinimidyl ester (PEG2000-SC, number-average molecular weight is 2,000) was purchased from Sinopeg Biotech Co., Ltd. (Xiamen, China). Nicotine, ethanol, and dopamine were purchased from TCI. Trypsin, penicillin, streptomycin, fetal bovine serum (FBS, Gibco), and Dulbecco’s Modified Eagle


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Medium (DMEM, high glucose) cell culture medium, FITC-labeled tyrosine hydroxylase antibody (TH-anti/FITC) and rat pheochromocytoma cell line (PC12) were purchased from KeyGen BioTECH. Methylthiazolyl-diphenyltetrazolium bromide (MTT) came from Beyotime Institute of Biotechnology. Male ICR mice were 6–8 weeks old and 18-22 g weight (Experimental Animal Center, Nanjing Medical University). The Maestro EX optical imaging system (Cambridge Research & Instrumentation) was used for living animal imaging. Synthesis of the conjugated polymer-g-PEG/PFBA (CP-EB): The synthesis route was shown in Figure S1. Compound 1 (0.22g), compound 2 (0.64g), and compound 3 (0.25g) were added to a degassed solution containing 25 mL of toluene and 12.5 mL of 2 mol/L potassium carbonate. Catalyzer tetrakis-(triphenylphosphine)-palladium (50 mg) was feed to the solution rapidly. The mixture reacted at 85-90°C for 48 hours in inert atmosphere. Then it underwent extraction by chloroform, washing by saturated salt water and then distilled water for three times. Precipitation from acetone provided conjugated polymer containing amine groups (CP-NH2) (brown solid, yield of 58%). Molecular weight was measured by gel permeation chromatograph (GPC, Agilent Technologies, calibrated by poly(ethylene glycol) standard; DMF as mobile phase). The numberaverage molecular weight is 8,700 g/mol; the polydispersity is 1.26. NMR spectra were obtained on Bruker DRX-300. Chemical shifts in CDCl3: 7.35-7.80 (m, Aromatic-H), 2.74 (s, pyrazinemethyl), 1.87-2.23 (m, fluorene-methylene, N-methylene), 0.70-0.95 (m, alkyl). CP-NH2 was subsequently reacted with 3-fluoro-4-carboxyphenylboronic acid (FPBA) and PEG2000-SC by the amidation reaction to afford CP-EB. CP-NH2 (0.16 g, 0.24 mmol, n (NH2)=0.48 mmol) and 4-dimethylaminopyridie (0.098 g, 0.08 mmol) were put into 30 mL of anhydrous DMSO containing FPBA (0.055 g, 0.30 mmol) and N, N’-dicyclohexylcarbodiimide (DCC) (0.062 g, 0.30 mmol) and reacted for 60 min at 0°C under an argon atmosphere. After


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reaction for 1 day at room temperature, excessive PEG2000-SC (0.18g, 0.09 mmol) was feed and continuous reacted for 3 days at room temperature. After stored in the refrigerator overnight, the precipitate was removed. The filtrate was dialyzed in the distilled water for 72 hours by dialysis membrane (8-10 KD), then freeze-dried to yield CP-EB (86%). Proton chemical shifts in CDCl3: 7.40-8.00 (m, Aromatic-H), 6.82 (s, amide), 3.54-3.68 (m, ether), 2.74 (s, pyrazine-methyl), 1.98-2.25 (m, Fluorene-methylene, N-methylene), 0.74-0.98 (m, alkyl). Boron-11 chemical shift in DMSO-d6 is 26.9. Preparation and test of the DRNs: The DRNs were synthesized by solvent exchange. CP-EB (2 mg) was dissolved in 1 mL of tetrahydrofuran (THF). Then distilled water (2 mL) was added to the THF solution by micro-injection pump at speed of 1 mL/h with stirring (850 rpm). The solution was then dialyzed in the distilled water by dialysis membrane (8-10 KD) for 24 h. The nanoparticle suspension was filtered by 0.22-micron membrane, and then stored at 4 °C. The hydrodynamic diameter and morphology of the nanoparticles were tested by dynamic light scattering (Malvern Mastersizer 2000) and TEM (JEM-1011). Absorption spectrum was recorded by UV-1800 (Mapada). Emission spectra were collected by FM-4NIR (Horiba Jobin Yvon). Two-photon absorption and fluorescence spectra were measured using optical multichannel analyzer (Princeton Acton SP2500i). The emission signals were collected upon excitation from 825 to 950 nm at 25 nm intervals. The images were collected from the slice incubated with the DRNs for 12h at 4oC with Olympus FV10-ASW two-photon fluorescent microscope. Fluorescence response properties of the DRNs: The fluorescence emission spectrum of 1 µM nanoparticles solution was measured. Then the solution was treated with dopamine of concentrations ranging from 10 nM to 10 µM. The spectra were collected after mixing for 10 min.


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Relative fluorescence intensity change at different concentrations of dopamine was analyzed. The fluorescence intensity changes of the DRNs after adding 10 µM other interferences, including tyrosine, tyramine, glucose, epinephrine, ascorbic acid, and norepinephrine, were also collected. The optical stability of the nanoparticles was studied in artificial cerebrospinal fluid (ACSF, composition in mM: NaCl 140, KCl 5, CaCl2 2, MgCl2 2, HEPES 10, D-glucose 10; pH 7.4), ACSF with 10% FBS, and ACSF under 12h white light irradiation (87mW/cm2). Cell culture and confocal fluorescence imaging: The PC12 cells were seeded in confocal microscope dishes at an intensity of 1×105/mL in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2. The cells were firstly incubated with FITC-labeled tyrosine hydroxylase antibodies (THanti/FITC) for 120min,42 and then they were washed twice with PBS and followed by incubating with the DRNs (5 µM) for 4h. The images of the co-stained cells were afforded by confocal laser microscope (Zeiss, Model LSM 710). The signals of the DRNs were collected at 650−750nm and excited at 595nm, and the fluorescence of tyrosine hydroxylase was collected at 500-600nm with excitation at 488nm. For detecting dopamine fluctuation in the PC12 cells, the cells were divided into two groups: one was incubated with DMEM and the other was stimulated by DMEM with 100 nM nicotine. Then, all of the cells were incubated with the DRNs (5 µM) for 4h. At last, the cells were washed twice by PBS. The images were collected by confocal laser microscope (Zeiss, Model LSM 710). Cytotoxicity Analysis: MTT assay determined cytotoxicity of the DRNs to the PC12 cells. The cells were seeded in a 96-well plate. The intensity was 0.5×104 cells per well in DMEM containing 10% FBS, as well as 1% penicillin/streptomycin at 37oC for 24h. Then, the medium was changed to DMEM supplemented with the DRNs in different concentrations for 24h. After


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that, the culture was replaced by 200 µL PBS, and 10 µL MTT (5 mg/mL) was added to each well and cultured for 4h. Then the solution was discarded, and 200 µL of DMSO was put into each well to dissolve the product. The absorbance was tested at 490 nm by microplate reader (Thermo Electron Corporation). The absorbance of the untreated cells and blank was taken as positive and negative control, respectively. In vivo observation for endogenous dopamine sensing in the brain: Male ICR mice were used for all animal experiments. Before surgery, the mice were weighed, anesthetized with isoflurane, and put into a stereotaxic apparatus. The cranium was exposed with a mid-sagittal incision. A hole was drilled with a dental drill mounted in the stereotaxic frame over the ventral tegmental area to the following coordinate: anteroposterior ±2 mm; lateral, ±2mm. A micro-injector containing the DRNs (10 µM) was lowered 3.0 mm to cerebral ventricle, and 20 µL of solution was injected in 2 min.

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Nicotine was chosen for activating mice and stimulus of dopamine enhancement in

the brain. The mice were treated with 0.1 mg/kg nicotine

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or 0.5g/kg alcohol by tail vein

injection. The NIRF images were collected for PBS-treated normal mice and the nicotineactivated mice. Furthermore, a seven-day period of nicotine treatment was performed to induce nicotine-addicted model in ICR mice. Then we tracked dopamine fluctuation in the nicotineaddicted mice with NIRF imaging. Spontaneous behavior tests: The spontaneous behavior test was carried out according to the standard method45, which was performed for accessing the effect of long-time nicotine exposure to the mice.46-47 The animals were tested between 14:00 and 16:00, with identical light and temperature in the chambers. Motor activity was collected in 1 hour, separated into three 20min spells, in a cage (40 cm*40 cm*25 cm) placed two series of horizontal infrared counters.


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Counting happened when mouse moved horizontally through infrared beams 1 cm above bedded floor. Statistical analysis：The data of fluorescence intensity change and spontaneous behaviors were subjected to Student’s t-test. 3. Results and discussion 3.1 DRNs fabrication and properties Neural activity imaging is critical for understanding how the brain produces consciousness and feelings, and potentially illuminating the causes of some major mental health disorders. To meet the requirements of minimal invasion and high spatial resolution for brain activity mapping, we successfully design the DRNs, which are capable of deep-tissue penetration and fluorescence imaging of brain (Figure 1a). The core of the DRNs consists of a hydrophobic π-conjugated polymer with alternatively copolymerized units of fluorene and dithiophene-thienopyrazine, where fluorene alone is high-efficient blue-emitting material and dithiophene-thienopyrazine is effective in shifting emission maximum of the conjugated polymer to near-infrared window. Due to their high photostability and biological compatibility, conjugated polymer nanoprobes have provided a competitive approach for high-resolution in vivo imaging and nanoscale 3D tracking cellular processes, such as drug or nucleic acid delivery.48-49 Realizing of brain activity mapping also urges the DRNs to have uniform size and high stability in aqueous solution. Therefore, flexible hydrophilic PEG side-chains are introduced to the rigid hydrophobic π-conjugated backbone. The amphiphilic copolymers are robust nanoparticles in water. The morphology of the nanoprobes is observed using transmission electron microscopy


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(TEM) (Figure 1b). The DRNs show average size of 100 nm (Figure 1c). Spherical shape of the nanoprobes in the TEM image is obviously identified. It is smaller than that in aqueous solution. It comes from the collapse of hydrophilic polymer layers when the nanoprobes are dried for observation in high vacuum environment. Absorption and fluorescence emission spectra of the DRNs in aqueous suspension are shown in Figure 1d. The nanoprobes have two absorptions at 410 and 580 nm. They originate from the πconjugated electron system. The maximal fluorescent peak is found at 720 nm, and the Stokes shift is 140 nm (λex=580 nm). The emission wavelength is in valid interrogation range for in vivo imaging with small interference by tissue auto-fluorescence.


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Figure 1. (a) Schematic illustration of preparing the DRNs and chemical structure of CP-EB. (b) TEM image and (c) hydrodynamic diameter distribution of the DRNs. Scale bar, 100 nm. (d) Normalized ultraviolet-visible absorption and fluorescence emission spectra of the DRNs in aqueous solution. The corresponding excitation wavelength is 580 nm.

3.2 In vitro fluorescence response of the DRNs The DRNs are sensitive to the concentration fluctuation of the neurotransmitter dopamine in physiological environment of brain. As presented in Figure 2a, the emission intensity at 720 nm from the DRNs (1 µM) in phosphate-buffered saline (PBS, pH 7.4) decreases distinctly when the dopamine concentration increases from 0 to 10 µM. A good linear correlation (R2=0.9949) is found between the emission intensity ratio (I0-I)/I0 (where I0 and I represent fluorescence maximum in the absence and presence of dopamine, respectively) of DRNs and logarithmic concentration of dopamine within 0.01-10 µM (Figure 2b). It indicates that the DRNs have the potential to realize high-sensitivity probing of the neurotransmitter both in vitro and in vivo. Each nanoprobe contains many PBA groups on the surface, which adsorb and enrich dopamine molecules surrounding it. This increases the chance of fluorescence quenching between the DRNs and dopamine via a photo-induced electron transfer process, which leads to their high sensitivity to the neurotransmitter. To identify the specific combination with dopamine, several other interfering substances in physiological conditions are introduced to the fluorescence sensing of the DRNs. They include tyrosine (TR, an important precursor to neurotransmitters), tyramine (TA, trace amine produced from tyrosine), glucose (Glc, most widely used sugar in living organisms with 1,2-diol group),


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ascorbic acid (AA, a molecule greatly restricts the selectivity in electro-analysis techniques with a oxidation potential to that of dopamine), and epinephrine/norepinephrine (EP/NE, other two catecholamines in human brain).50-51 As shown in Figure 2c, dramatic fluorescence change of the DRNs is observed for dopamine with a concentration of 1 µΜ. The interferences (10 µM) only lead to small fluorescence changes, and they have little influence in the physiological condition. For the DRNs, dopamine-selectivity in the presence of competing molecules, i.e. EP (1 µM), L-DOPA (1µM), and DOPAC (1µM), is also confirmed (Figure S2). L-DOPA and DOPAC are the precursor and metabolite of the neurotransmitter, respectively. Most fluorescence sensors present a selectivity between dopamine and ascorbic acid or uric acid.52 The DNA-wrapped carbon nanotube has high selectivity between dopamine and norepinephrine or epinephrine.21 The binding capacities of PBA-functionalized polymer microspheres in HEPES (pH 8.5) are 398, 38, and 47 µmol/g for dopamine, epinephrine, and norepinephrine, respectively.53 Thus, the affinity of epinephrine or norepinephrine is significantly lower than that of dopamine. Acid dissociation of PBA or diol is detrimental to their binding affinity. Acid dissociation constants are 10-8.9, 10-8.66, and 10-8.64 for dopamine, epinephrine, and norepinephrine, respectively. As a result, dopamine has the largest binding affinity. The equilibrium constant for dopamine binding is about 3*109. High binding affinity of dopamine may also arise from dynamic effect. PBA-functionalized molecularly imprinted polymers show extremely high affinity to dopamine in the presence of epinephrine and norepinephrine.54 It is postulated to rapid adsorption dynamics of the dopamine molecules on specific surface binding sites. The DRNs are spherical nanoparticles with about 105 functional groups on each nanoparticle. As a result, stereo-hindrance effect may have influence on the


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adsorption dynamics. Dopamine molecules need the smallest space for adsorption, leading to high affinity to the DRNs. The stability of the DRNs in physiological environment is also taken into account. Figure 2d shows that the fluorescence intensity is very stable in the artificial cerebrospinal fluid (ACSF). When the DRNs are in the ACSF with 10% fetal bovine serum (FBS), no obvious emission intensity change is observed. Then, the DRNs in ACSF are upon irradiation under white light (87mW/cm2) for 12 hours. They can still work well as the near-infrared emissive nanoprobes. It indicates that the DRNs have high photo-stability and remain steady in the physiological environment of brain, which is vital to long-term and dynamic fluorescence imaging.

Figure 2. (a) Fluorescence spectra of the DRNs (1 µM) in PBS (20 mM, pH 7.4) obtained upon treated with dopamine with the concentration from 0 to 10 µM. (b) The relationship between the


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emission intensity ratio (I0-I)/I0 of the DRNs and logarithmic concentration of dopamine. (c) The emission intensity change (∆I) of the DRNs nanoparticles (1 µM) in PBS buffer (20 mM, pH 7.40) in the presence of dopamine (DA) (1 µM), tyrosine (TR) (10 µM), tyramine (TA) (10 µM), glucose (Glc) (10 µM), epinephrine (EP) (10µM), ascorbic acid (AA) (10 µM), and norepinephrine (NE) (10 µM). (d) Stability of the DRNs in the physiological environment and upon irradiation under white light (87mW/cm2) for 12 hours. 3.3 The DRNs for Cellular Fluorescence Imaging PC12 cells are chosen as dopaminergic neuron model to evaluate the intracellular performance of the DRNs in imaging and sensing the endogenous neurotransmitter dopamine. The cells can explore the Parkinson's disease with respect to molecular/neurochemical factors. Tyrosine hydroxylase is an important enzyme for dopamine synthesis.55 To identify intracellular location of the DRNs, the cells are co-stained with FITC-labeled tyrosine hydroxylase antibody (THanti/FITC) and the nanoparticles, respectively. As shown in Figure 3a, the fluorescence emissions of DRNs (red) are located in the cytoplasm; and TH-anti/FITC (green) is targeted to neuronal cell membrane and cytoplasm. The fluorescence signals of the DRNs coincide well with the TH-anti/FITC signals in the cytoplasm (yellow), indicating that the DRNs can map the endogenous neurotransmitter with high spatial resolution in living cells. Nicotine is an addictive drug that increases levels of neurotransmitter dopamine in the reward pathways, i.e. the brain circuitry that regulates feelings of pleasure. Herein, fluorescence images of the DRNs in the living cells under a 100 nM nicotine stimulation for 30 min are collected by confocal laser microscope. Compared with the control group, a remarkable decrease of fluorescence signal of the DRNs is observed under nicotine-activated condition (Figure 3b). The


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mean emission density of the DRNs in the cells have decreased about 30% of the original value after nicotine stimulation (Figure 3c). It meets well with the common sense that nicotine can induce cellular release of dopamine, and demonstrates that the DRNs can dynamically monitor the change of the neurotransmitter level in the living cells. Biocompatibility of the DRNs is also important for practical application in intracellular system and brain. Cell viability is verified by the MTT assay (Figure 3d). The DRNs present insignificant cytotoxicity to the cells within the concentrations used in this study. This agrees with the general believing that conjugated polymer nanoparticles are highly biocompatible. The PEGylation, i.e. covalently attached PEG layers on the surface of the DRNs provides their water solubility, and may also "mask" them from the immune system.


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Figure 3. (a) Confocal laser scanning microscopy images of the PC12 cells seeded with the DRNs and FITC-labelled tyrosine hydroxylase antibodies (TH-anti/FITC). (b) Fluorescence images of the cells seeded with 5 µM the DRNs. Both the control and 100 nM nicotine-activated cells are shown. (**, P൏0.01) (c) Emission mean density of the DRNs in the cells with or without nicotine stimulus. (d) Cell viability treated by the DRNs at various concentrations for 24 hours. 3.4 NIRF imaging of the drug addiction in mice brain Drug addiction has enduring effects on brain circuits and human behavior. 56 The association of the neurotransmitter dopamine with the reinforcing effects or abuse of drugs is well recognized.


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Dynamically mapping neural activities in living brains will help understand the formation

process of drug addictions and its influence on brain. NIRF imaging can dynamically track the change of the neurotransmitter level in the brains of mice evoked by nicotine or alcohol, and determine their influence on neural activities. Experimental scheme is shown in Figure 4a. The DRNs are intracranial injected into mid-brain as near-infrared fluorescent nanoprobes for in vivo detection and tracking of neurotransmitter in living mice. Three-month survival rate of the mice after injection is 100%. The fluorescence signals focus on the mid-brain, where the ventral tegmental area is located (Figure 4b). VTA is a source region of dopaminergic cells.

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The

average initial fluorescence signals of the normal and nicotine treated groups are 0.00035 and 0.00032 scaled counts/s, respectively. The mean fluorescence densities of the DRNs in mid-brain of the nicotine-activated mice and normal group with PBS injection are also shown. Relatively weak fluorescence intensity of the DRNs is observed in the nicotine-activated groups after 30 minutes of the injection of nicotine through cauda vein, while the fluorescence intensity of the normal group keeps stable. It suggests that the level of the neurotransmitter has increased in the mid-brain after the mouse is stimulated by nicotine, because this brain region largely involves in the drug and natural reward pathway. Nicotine as an addictive could activate the reward pathway and bring strong happy feelings to the brain. Excessive alcohol consumption can cause structural and functional abnormalities of the brain. It could lead to regional brain damage and cognitive dysfunction, and eventually increase risks of health, social, and economic problems. Imaging the neural activities in vivo when stimulated by alcohol will help figuring out how alcohol abuse can shape the brains. The activated group is injected alcohol (0.5 g/kg) through cauda vein as the stimulation, and the normal group is


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injected PBS buffer. After 1 hour, the control and alcohol-activated groups are sacrificed. The brains are isolated for fluorescence imaging (Figure 4c).

Figure 4. (a) Schematic illustration experimental approach. The DRNs are injected to mid-brain of the mouse. (b) Left: In vivo NIRF brain imaging of the normal and the nicotine-activated mouse before and after nicotine injection. Right: Fluorescence emission mean density of the DRNs in the normal and the nicotine-activated mouse. (c) Ex vivo NIRF brain imaging and


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fluorescence emission mean density of the normal and alcohol-activated mice (the injection site is located in the dashed line cross the brain median line). (*, P൏0.05), n=5 animals per group. The fluorescence signals mainly locate in the mid-brain. Compared to the normal group, the alcohol-activated group shows obvious weak fluorescence from the DRNs in the mid-brain, indicating high neurotransmitter level. Mid-brain is an important part of the reward pathway, and prefrontal cortex is implicated in decision making and moderating social behavior. Thus, alcohol as an addictive could interact with the reward pathway in the brain. Excessive alcohol consumption could affect the decision making process, which may lead to severe injuries, violence and traffic accidents. The drug-induced change of dopamine levels in the brains has been studied earlier.

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Dopamine level change in the mice brain after nicotine or alcohol stimulation is measured by high-performance liquid chromatography (HPLC) (Figure S6). The mice were divided into three groups, the normal, nicotine-activated, and alcohol-activated. The brain tissue was prepared according to the reference62. The dopamine contents in the brain after nicotine and alcohol treatments increase 2.1 and 2.5 µg/g, respectively. The results are consistent with those observed by brain fluorescence imaging. The mouse injected with the DRNs was treated with reserpine (5 mg/kg), which leads to depletion of neurotransmitter dopamine, for 2 hours. The NIRF images before and after treatment were collected. As shown in Figure S8, in the presence of the reserpine, the dopamine level falls down, and consequently the fluorescence intensity of the DRNs increases. We then investigate the brain activity in the nicotine-addicted mice. The experimental scheme of the spontaneous behavior test is shown in Figure 5a. Counting happens as the mouse passes low-


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level grid of infrared beams. Total activity of the test mice is measured during 60 minutes, which are separated into three spells. The mice addicted to nicotine are significantly more active in total activity during the first 20-min spell than the normal ones (Figure 5b). During the second and third 20-min spells, the addicted mice have similar motion with the normal ones. Then, the brain activity of the normal and addicted mice is analyzed by NIRF imaging with the DRNs. As shown in Figure 5c, the fluorescence of the DRNs in the nicotine-addicted mice is weaker than that in the normal. The NIRF brain imaging with the DRNs could reflect the brain activity level in living mice brain, and the results meet well with the behavior of the mice. Quantification analysis result at t=0 is shown in Figure S10.

Figure 5. (a) Schematic illustration of spontaneous behavior test in the normal and nicotineaddicted mice. (b) Spontaneous behavior of the normal and addicted mice in 0-20, 20-40, and 4060 minutes. (c) In vivo NIRF images about brain activity of the normal and addicted mice. (d) Schematic illustration of the experimental approach for stimulating the addicted mice. The DRNs


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are injected to mid-brain of the addicted mouse. (e) In vivo NIRF brain images of the nicotineaddicted mice at different time points after nicotine injection. (f) Fluorescence emission mean density at different time points of the normal mice without treatment and nicotine-addicted mice treated with 0.1mg/kg nicotine. (*，P൏0.05), n=5 animals per group. Then we observe brain activity of the nicotine-addicted mice when they are injected with same dose of nicotine used in the inducing process. NIRF imaging of the nicotine-addicted mice brain is performed after stimulation (Figure 5d). There is little fluorescence intensity change in 30 minutes after the injection of nicotine (Figure 5e and f). The reason may be that after the longterm drug use, higher concentration of nicotine is needed to activate the reward pathway. The addicts might ask for higher intakes and a faster delivery to brain. This leads to the excessive usage of the drugs and makes addiction hard to quit and easy to relapse.

4. Conclusions In summary, we have developed functional NIRF nanoprobes for tracking neural activity both in vitro and in vivo. We track dynamical change of the dopamine level in physiological environment and the releasing of the neurotransmitter in living dopaminergic cells in response to nicotine stimulation. NIRF imaging can dynamically track the neurotransmitter levels in the mice mid-brain activated by stimulants and evaluate the long-term effect of addictive substances to the brain. This strategy could evaluate the process of drug addiction and has the potential for treating drug abuse/addiction. The functional NIRF imaging also opens unique window to study neural activity in physiological condition.


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The synthesis route, dopamine-selectivity in the presence of interferences, dopamine dynamic response in solution, bio-distribution of the nanoparticles in brain, dopamine level change in response to nicotine or alcohol stimulation in brain, impact of nicotine on sensor fluorescence, two-photon properties of DRNs.

AUTHOR INFORMATION Corresponding Author * Corresponding author: E-mail: [email protected], Phone: 86-25-89687807. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. #These authors contributed equally. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (No. 21174060), Innovation Project of Jiangsu Province (No. KYZZ16_0042), as well as the Program for Innovative Research Team in University.


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Near-Infrared Fluorescent Nanoprobes for Revealing the Role of

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