Mitochondrial sensitive probe with aggregation‐induced emission characteristics for early brain diagnosis of Parkinson's disease

The early diagnosis of Parkinson's disease (PD) provides opportunities for early intervention to slow the progression of neurological degeneration in patients, particularly as the aging population increases in our society. Among a series of pathological features of PD, mitochondria abnormalities have been identified as central event that occurs at the early stage of PD. However, the method for detecting mitochondrial abnormalities‐associated early PD has not been fully developed. We herein report a specifically mitochondrial targeting probe (named TPA‐BT‐SCP) that is able to characterize mitochondria abnormalities for early diagnosis of PD and monitor PD neurodegenerative progress. The probe is an aggregation‐induced emission (AIE) probe with a strong positive charge, a 3D distorted molecular structure, and a separated HOMO‐LUMO distribution, designed with unique molecular design guidelines. Our research demonstrated that TPA‐BT‐SCP could emit stable and strong fluorescence, and rapidly accumulate in mitochondria due to the negative charge. After intranasal administration of 1‐methy‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced PD mice, TPA‐BT‐SCP successfully bypassed the blood−brain barrier to light up the brain, allowing the grading of PD severity based on its high sensitivity. Taken together, this work develops a novel AIE probe that exhibits dramatically high sensitivity to mitochondrial changes and enables noninvasive diagnosis of early PD in the brain.

[7] Clinical evidence indicates that early intervention in PD using disease-modifying medication such as monoamine oxidase-B inhibitors combined with tocopherol (the DATATOP trial) and rasagiline (the ADAGIO trial), or physical activity, effectively delays the progression of PD. [8][9][10][11] Therefore, in order to provide early treatment for PD patients, developing a reliable early diagnostic method for PD is of great significance, reducing complications and promoting functional recovery in patients.
[17][18] Sporadic PD, which accounts for over 90% of PD cases, has a higher mitochondrial density in dopaminergic neurons of SN compared to other neuron types. [19]As the disease progresses, the quality and function of mitochondria in dopaminergic neurons will decline, causing mitochondrial dysfunction. [20,21]Therefore, mitochondrial dysfunction serves as a universal indicator that could be used at the early stage of PD, contributing to grading the severity of PD neurodegeneration.
Recent studies have focused on developing methods for the early diagnosis of PD.Several fluorescent probes, such as the targeted probe NUU-1 for HClO and the two-photon probe U1 for detecting monoamine oxidase B activity, are mainly suitable for established PD models. [22,23]Small molecule probes containing lipophilic cations, such as Rhodamine 123 or Mito-Tracker FM dyes, are commonly used mitochondrial fluorescent probes.[26] To overcome these limitations above, probes with aggregation-induced emission (AIE) characteristics have been developed, which have attractive characteristics in fluorescence imaging, including high emission brightness in aggregates, a high photobleaching threshold, and great tolerance at any concentration. [27]Miao et al. constructed near-infrared II fluorescent probes with a donor-π-acceptor (D-π-A) structure for specific detection of Aβ plaques in an Alzheimer's disease model mouse. [28]ue et al. reported an AIE probe activated by nitric oxide, which enables the detection of brain inflammation in vivo by modulating molecular geometry and energy conversion processes. [29]Therefore, the structural properties of AIE are suitable for high-sensitivity and high-fluorescence-intensity imaging of mitochondria in the brain.
In this study, we developed a novel mitochondrial-sensitive AIE probe (named TPA-BT-SCP) containing triphenylamine (TPA) as the electron donor and pyridinium salts as the electron acceptor for mitochondria-targeted imaging and in vivo detection of PD development.The probe has a strong positive charge, which allows it to selectively aggregate in negatively charged mitochondria and turn on its fluorescence, making it highly suitable for bioimaging applications.To verify its potential for early diagnosis and severity grading of PD, we constructed a classic PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection for 1-7 days and graded the progression of the disease based on mouse behavioral detection and the number of dopaminergic neurons lost in the SN.This approach simulated the preclinical (3 days of injection) and early (5 days of injection) stages of clinical PD. [30][31][32][33] The results showed that the TPA-BT-SCP probe rapidly and sensitively targeted mitochondria to achieve the grading of MPTP-induced PD with different severities.When administered intranasally, the positively charged probe accumulated on the negatively charged nasal mucosa, enabling it to diffuse into the interstitial fluid and reach the olfactory and trigeminal nerve pathways, ultimately penetrating the brain parenchyma. [34]e probe could be rapidly detected within 30 min after noninvasive intranasal instillation.These findings provide a new possibility for early diagnosis of PD and greatly expand the biomedical applications of AIE in advanced brain disease diagnosis and precision medicine.

Design and synthesis of TPA-BT-SCP
As shown in Figure 1A, a mitochondrial molecular probe with D-π-A conformation and rich intramolecular motion units was designed and synthesized.To create an excellent AIE effect, TPA was used as a donor unit with strong electron properties.As a π-bridge unit, the planar benzothiadiazole (BT) ring can narrow the probe HOMO-LUMO gap and effectively redshift the absorption spectra of the dye. [35]Pyridinium salts (SCP) target the mitochondria as the anchoring part of the core. [36]Here, a D-π-A-type probe (TPA-BT-SCP) targeting mitochondria was designed and synthesized.
It targets the mitochondrial membrane by charge-coupled interaction.The on or off state of the fluorescence produced by AIE changes according to changes in mitochondrial membrane potential.The intermediates and the final product have been characterized by nuclear magnetic resonance (NMR) and high-resolution mass spectrum (HRMS) (Figures S1-S6).

Photophysical properties of TPA-BT-SCP
Further structural information on the TPA-BT-SCP was explored by first-principles density functional theory calculations.The TPA-BT-SCP, as shown in Figure 1B,C, exhibited a certain degree of LUMO-HOMO separation, forming an energy gap of 1.88 eV, which allowed for a strong absorption/emission band in the redshifted wavelength region.We then investigated the photophysical properties of TPA-BT-SCP in the tetrahydrofuran (THF)/water system.As shown in Figure 1D, the photoluminescence (PL) intensity was very weak and remained stable when the water percentage (fw) was less than 70%.A further increase in the fw resulted in a significant increase in the fluorescence intensity of TPA-BT-SCP due to the formation of aggregates, which indicated that TPA-BT-SCP is an AIE-active fluorescent probe.The ultraviolet-visible (UV-vis) absorption spectrum showed that the maximum absorption of TPA-BT-SCP in the dispersed state (pure THF) is at 520 nm (Figure 1E) and that in the aggregate state (Water: DMSO = 99:1) is at 510 nm (Figure 1F).These results demonstrate that TPA-BT-SCP has significant AIE features.

In vitro mitochondrial response of TPA-BT-SCP
The biocompatibility of a fluorescent probe is crucial for disease early diagnosis.We thus selected differentiated SH-SY5Y cells to simulate neuronal properties for subsequent in vitro experiments.Before in vitro PD model induction, we first determined a noncytotoxic range of 1-2.5 μM for SH-SY5Y cells from TPA-BT-SCP (Figure S7A).Furthermore, we assessed whether the TPA-BT-SCP probe impacted on mitochondrial function by measuring the energy production adenosine triphosphate (ATP) content of mitochondria in SH-SY5Y cells, compared with that in untreated cells, which demonstrated that the probe did not affect mitochondrial function (Figure S7B).After verification of low cytotoxicity, SH-SY5Y cells were incubated with TPA-BT-SCP for 4 h.TPA-BT-SCP was specifically distributed in the mitochondria of SH-SY5Y cells and emitted intense red fluorescence, which was confirmed by the excellent fluorescence overlap with the commercially available mitochondria-targeted dye Mito-tracker Green (Figure S7C,D).Then, colocalization experiments with Mito-tracker green were conducted and a good Pearson's correlation coefficient (PCC) of 0.88-0.91 was calculated, indicating that TPA-BT-SCP can be located in the mitochondria of SH-SY5Y cells with excellent selectivity (Figure S7E).
To assess the early diagnostic effect of TPA-BT-SCP on PD in vitro, we generated PD cell models with different concentrations (0, 50, 100, 150, and 250 μM) of 1-methyl-4phenylpyridinium (MPP + ) to guide distinct stages. [37]MPP + is a well-known drug for in vitro PD modeling that induce mitochondrial damage and cause neuronal toxicity.This probe observed a significant fluorescence signal in the cytoplasm of SH-SY5Y cells at a lower concentration (2.5 μM) and created a network structure in comparison to the probe (5 μM) that specifically targeted the mitochondria of cancer cells in previous investigations. [27]Our results showed that the fluorescence signal of TPA-BT-SCP significantly increases in nerve cells (Figure 2A).Due to the AIE nature of this probe compared to the Mito-tracker, the intracellular fluorescence intensity in the cell increases with the accumulation of TPA-BT-SCP and can be easily distinguished.Note that the fluorescence signal intensity of the probe displayed a linear downward trend with increasing MPP + concentration due to mitochondrial membrane potential loss caused by mitochondrial damage (Figure 2A,B). [38,39]The colocalization experiment of TPA-BT-SCP and Mito-tracker still had a decent PCC in PD cell models (PCC = 0.89-0.96)(Figure 2C), indicating that TPA-BT-SCP had excellent mitochondrial targeting ability throughout, which should be attributed to the pyridinium group.These results indicated that TPA-BT-SCP at low concentrations was highly sensitive to the detection of the fluorescence turn-on mode in mitochondria, demonstrating pronounced fluorescence variations in the early stage of mitochondrial damage.

Metabolism and accumulation of TPA-BT-SCP in mice brain
The sensitivity and accuracy of the probe for mitochondrial targeting in vitro give us confidence for the further investigation of early diagnosis of PD in vivo.In the initial phase, we administered the probe specifically into the SN region of the brain in healthy mice.This approach is employed to minimize probe interference within the in vivo circulation and facilitates the prompt and precise assessment of the probe's localization, metabolism, and aggregation within intracellular mitochondria of the living brain (Figure S8).Then, to evaluate the metabolic duration of the probe, we measured the fluorescence intensity of the probe at different time points (Figure S9A).According to the statistics, the probe could essentially be metabolized 96 h after administration (Figure S9B).Rapid fluorescence imaging was a big advantage of real-time detection, so we fluorescently photographed the accumulation of probes in the mouse brain at different time points (0, 1, 2, 4, 8, and 12 h) (Figure S10A).According to the real-time monitoring data, the fluorescence intensity increased between 1 and 4 h after the probe administration and peaked at 4 h (Figure S10B).These results indicate that the probe accumulated in the neuronal cells of the SN brain area in a short time, which meets the requirements of rapid detection in situ.

Early PD diagnosis in vivo
In order to thoroughly investigate the applicability of our TPA-BT-SCP for PD evaluation, MPTP was given intraperitoneally at different time intervals to represent the preclinical stage to the late stage of PD in mice brains. [40,41]The preclinical model (3 days of intraperitoneal injection of MPTP) showed that less than 50% of dopaminergic neurons in the SN degenerate in the absence of motor defects. [42]he early symptomatic period model (5 days of intraperitoneal injection of MPTP) demonstrated that approximately 50% of dopaminergic neurons in the SN degenerate in the presence of mild motor deficits. [4]After 7 days of intraperitoneal injection of MPTP, the motor coordination of the mice dropped dramatically, reaching the late stage of PD (Figures 3A-D and Figure S11A).These results indicate that the MPTP model effectively represents PD pathogenesis from the onset of preclinical stage to the late stage.Next, we investigate the effect of the probe on the early detection of PD, the mice at different stages of PD were determined 4 h after the TPA-BT-SCP probe administration.Encouragingly, the fluorescence intensity in the brain continued to decrease with increasing days of MPTP injections (Figure 3E).The statistical analysis showed that the fluorescence signal started to significantly reduce in the preclinical stage of PD (3 days) (Figure 3F), which is due to the high sensitivity of the probe to mitochondria.It was known that the loss of dopaminergic neurons in the SN brain region is a prerequisite for the induction of PDrelated motor symptoms.In addition, mitochondrial protein import was impaired in early-stage PD, which was related to the downregulation of translocase of the inner mem-brane 23 (Tim23), a key component related to mitochondrial dysfunction. [43]To further investigate the specificity of our probe to mitochondria in the progression of PD, we evaluated the fluorescent changes of the probe that costained with Tim23 and TH, an enzyme that converts L-tyrosine into a dopamine precursor, determining the degenerative neurons on PD (Figure 3G).By observing the representative images, we found that the fluorescent intensities of the TPA-BT-SCP probe in brains consistently intensify with the colocalized TH-labeled dopaminergic neurons with Tim23-labeled mitochondria in the SN region, which gradually decreased with increasing days of MPTP injection (Figure S11).Moreover, the PCC between the fluorescence intensity of the probe and Tim23-labeled mitochondria was 0.8719, which further confirmed that the TPA-BT-SCP probe can be used for accurate detection of early-stage PD by targeting mitochondria, providing broad prospects for understanding disease progression and drug screening (Figure 3H).

Noninvasive early diagnosis of PD in vivo
In light of the specificity, sensitivity, and quick response of our TPA-BT-SCP probe in the PD mouse model, we further explore other noninvasive administration forms to extend the application of the probe potentially in the clinic.One promising approach for drug delivery to the brain is intranasal administration.From a clinical perspective, intranasal drug delivery offers several advantages, such as being noninvasive, easily accessible, and patient compliance. [44][47][48][49] We first assessed the ex vivo organ distribution of the probe using optical imaging and calculated the accumulation in the main organs.Mice were intranasally administered with 10 μL of 2.5 μM TPA-BT-SCP.Consistent with the ex vivo imaging, the results of injected doses of tissue showed that the probe reached its peak accumulation in the brain at 0.5 h, but started to decrease after 1 h.After 4 h, only a minimal amount of the probe remained in the brain tissue, which was not apparently visible in the collective images.This may be attributed to the brain being a highly perfused organ with a fast blood flow rate. [44]Consequently, the probe is likely to rapidly traverse through the brain tissue, reducing its residence time in the brain.Furthermore, the probe primarily accumulated in the liver and lungs and was cleared from the body through hepatic and pulmonary routes (Figure S12A,B).
To verify whether the probe could grade the severity of PD in mice through intranasal administration, we instilled TPA-BT-SCP into the nasal cavity of PD model mice (Figure 4A).After 30 min, the brain imaging was performed, which quickly displayed a pronounced signal in vivo.Representative image of the whole mouse illuminates that the probe could effectively image different degrees of PD after intranasal administration, and in vivo investigations evaluated that the TPA-BT-SCP administered in the nasal cavity still maintains great responsiveness to mitochondria (Figure 4B,C).Biosafety assessment is crucial for in vivo studies.Therefore, we next evaluate the potential effects of the probe TPA-BT-SCP on the major organs of mice.To ensure a comprehensive biosafety assessment, we examined the ATP content in mitochondria of each organ relative to untreated mice 24 h after intranasal administration of the TPA-BT-SCP probe.The results indicated that the probe does not interfere with mitochondrial function in the tissues (Figure S13A).Additionally, we conducted a hematoxylin and eosin (H&E) analysis on the major organs (Figure S13B).By comparing the histological features and cellular structures between the TPA-BT-SCP-treated and control groups, we confirmed that the probe did not induce toxic effects on major organs.These results illustrated that TPA-BT-SCP rapidly grades PD in a noninvasive manner, greatly expanding its applicability in clinical early diagnosis of PD.
In this work, we develop a mitochondrial sensitive probe for quickly noninvasive in vivo detection of early PD.To precisely target the negatively charged mitochondrial membrane and activate the AIE effect, we designed and synthesized a molecular probe with a strong positive charge that selectively binds to the negatively charged (−250 mV) mitochondria (Figure 5A,B).PD symptoms are exacerbated by mitochondria-related alterations, including disrupted mitochondrial membrane potential. [50]At this point, the probe targeted the mitochondria, and the AIE effect is turned on.Additionally, the probe not only exhibits high selectivity toward mitochondria in vitro but also possesses enable excellent signal output in vivo.After molecular targeted aggregation, the energy shift in the excited state further promotes the active intramolecular motion, which in turn boosts the in vivo AIE efficacy with a high signal-to-noise ratio.Therefore, the probe has been used for noninvasive mitochondrial imaging in early PD and defines the severity of the disease (Figure 5C).Intranasal injection, the noninvasive delivery with topical administration, helps to produce a fast and strong fluorescence signal in the brain, because the probe could be used for accurately detecting the mitochondriaassociated PD pathology with high selectivity and sensitivity in living mice.

CONCLUSION
In this study, we introduce a novel PD early detection probe with a strong AIE effect and the ability to precisely reflect the changes of mitochondrial membrane potential in neurons.The main function of mitochondria is to generate the energy necessary to power cells.Because of the high energy demands of dopaminergic neurons in the SN, these cells typically contain a higher number of mitochondria compared to other neurons.As a result, the SN is one of the brain regions with relatively higher mitochondrial levels. [21,51]hrough charge-coupled interaction, the positively charged TPA-BT-SCP probe is more likely to selectively accumulate in the negatively charged mitochondrial membrane of the SN, which give us an opportunity to exactly apply the TPA-BT-SCP probe for PD early diagnosis.Compare to the commercial mitochondrial probes, our mitochondrial sensitive probe TPA-BT-SCP exhibits improved sensitivity and robustness due to the advantages in terms of specific mitochondrial targeting, 3D distorted molecular structure, and separated HOMO-LUMO distribution with unique molecular design guidelines.These pronounced properties motivate us to further evaluate the probe for early PD diagnosis.Furthermore, this study also shows the link between mitochondrial damage and PD progression from the in vitro and in vivo tests.
Our study also investigated the application of the probe delivery to the brain through intranasal instillation.In nasal drug delivery, the brain and nasal cavity are interconnected through the olfactory pathway and peripheral circulation. [34]nce the drug is administered into the nasal cavity, it first comes into contact with the nasal mucosa.The nasal mucosa has high permeability and contains abundant blood capillaries. [52]Within the nasal mucosa, there are two pathways: the respiratory mucosal pathway and the olfactory mucosal pathway.When the drug enters the respiratory mucosal pathway, it may enter the systemic circulation and also be directly transported to brain tissue via the trigeminal nerve pathway.On the other hand, in the olfactory mucosal pathway region, the drug is directly delivered through the olfactory route or reaches the brain through diffusion. [53,54]he intranasal instillation offers the advantages of noninvasiveness, and self-administration, allowing the drug to bypass the blood−brain barrier by migrating from the nasal mucosa. [55,56]However, nasal drug delivery is subject to certain limitations.The nasal mucosa possesses well-developed epithelial cilia, which move toward the throat, effectively clearing substances bound in mucus out of the body. [57]To overcome this limitation, prolonging the residence time of the drug in the nasal cavity may increase the chances of drug absorption and delivery to the brain (Figure S14). [58,59]revious studies have indicated that the ability of small molecules to penetrate the nasal mucus is dependent not only on the size but primarily, on their surface. [60]Under the physiological conditions, the positively charged TPA-BT-SCP probes are apt to stick to the negatively charged mucinous protein, greatly slowing down the ciliary clearance of TPA-BT-SCP and quickly promoting its rapid accumulation in the brain within 30 min.
In conclusion, we have developed a mitochondria-targeted probe, TPA-BT-SCP, which offers a fast, convenient, and sensitive tool for early diagnosis of PD through noninvasive administration and rapid detection.This work will inspire further research into the use of sensitive bioprobes for early brain disease diagnosis, providing new perspectives for efficient probe development and enabling noninvasive, precise early biomedical imaging of the brain with significant potential for practical applications.Overall, early diagnosis is crucial for the management and treatment of PD, as it contributes to improving patients' quality of life and provides researchers with greater opportunities to enhance disease management and future therapies.

Reagents
All the chemicals were obtained from Sigma-Aldrich unless otherwise specified and used as received without further purification.The solvents for chemical reactions were distilled before use.Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation).

Instrumentation
1 H and 13 C NMR spectra were recorded on a Bruker-DPX 400 spectrometer.Chemical shifts are reported in ppm from tetramethylsilane with solvent resonance as the internal standard.HRMS were recorded on a Varian 7.0T FTMS Mass Spectrometer System operating in Matrix-Assisted Laser Desorption/Ionization Time of Flight mode.UV-vis absorption spectra were recorded on a Shimadzu UV-1700 spectrometer.Photoluminescence and afterglow spectra were recorded on a Perkin-Elmer LS 55 spectrofluorometer.The observation of nanoparticle morphology was investigated using transmission electron microscopy (TEM, JEM-2010FJEOL).Afterglow images and NIR fluorescent images were acquired using the Xenogen IVIS® Lumina II system under bioluminescence and fluorescence modes, respectively.Structures of DPA-TPE-DCM were obtained using the Gaussian 09 W program at the B3LYP/6-31G(d) level, Gaussian 09 program.

Synthesis of TPA-BT-SCP
Under an atmosphere of nitrogen, TPA-BT (407 mg, 1 mmol), cyano-Ph-Py (194 mg, 1 mmol), and EtONa (4 mg, 0.06 mmol) were dissolved in EtOH (5 mL), then the reaction solution was refluxed at 8 h.The crude product was purified by column chromatography on silica gel (PE/DCM = 3/1).Under an atmosphere of nitrogen, iodomethane (675 mg, 4.75 mmol) was added to a red solid (551 mg, 0.94 mmol) in acetonitrile (5 mL), then the reaction solution was refluxed overnight.The reaction was monitored by thin layer chromatography (TLC).Then, the solvent was evaporated under reduced pressure.The crude product was purified by column chromatography on silica gel (DCM/MeOH = 10/1).After completion, the reaction mixture was diluted with acetone (10 mL) and added to aq KPF 6 (15 mL).The mixture was stirred at 3 h.The organic phase was dried over with Na 2 SO 4 and concentrated under reduced pressure.The residue was gained as a red solid (635 mg, 85%).

4.6
Cell culture and differentiation SH-SY5Y cells were maintained in DMEM nutrient media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (100 U mL −1 penicillium and 100 μg mL −1 streptomycin) at 37 • C with 95% relative humidity and 5% CO 2 .SH-SY5Y cells were differentiated into mature neuronal phenotype by exposure to 10 μM retinoic acid for 3 days.

Cytotoxicity study
3-(4,5-Dimethyl-2-thiazolyl)−2,5-diphenyl-tetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of TPA-BT-SCP probes.SH-SY5Y cells were harvested in a logarithmic growth phase and seeded in 96-well plates (2 × 10 4 cells per well with 100 μL suspension) and grew to ∼80% confluence.Then, the culture medium was replaced with 100 μL fresh culture medium containing TPA-BT-SCP probes with various concentrations (the concentrations based on TPA-BT-SCP were 0, 1.25, 2.5, 5, and 10 μM, respectively).After incubating for 24 h, the culture medium was removed and the wells were washed three times with phosphate buffer saline (PBS), and 100 μL MTT dissolved in serum-free culture medium (0.5 mg mL −1 ) was added into each well.After 4 h, the MTT solution was removed cautiously and 150 μL dimethyl sulfoxide (DMSO) was added into every well.Following gently shaking for 10 min, the absorbance of MTT at 490 nm was measured by a Bio-Rad 680 microplate reader to evaluate the cell viability.

ATP assay
ATP content in animal tissues and SH-SY5Y cells was detected with the ATP assay kit (Beyotime Biotechnology) following the manufacturer's directions.SH-SY5Y cells were cultured in 6-well plates, treated with 2.5 μM TPA-BT-SCP, and incubated for 24 h.The medium was discarded after incubation and placed with 200 μL lysate to lyse the cells.
Repeatedly pipetting or shaking the culture plate when lysing the cells to make the lysate fully contact and lyse the cells.After lysis, cell lysates were centrifuged at 12,000 g at 4 • C for 5 min, and the supernatant was collected for subsequent determination.For tissue samples, the SN, lung, and liver were collected separately from mice 24 h after intranasal administration.Following a ratio of approximately 200 μL of lysis buffer per 20 mg of tissue, the tissue samples were mixed with the lysis buffer.Homogenization was then performed using a glass homogenizer or other homogenizing devices.Thorough homogenization ensures complete tissue lysis.After lysis, the mixture was centrifuged at 4 • C and 12,000 g for 5 min, and the supernatant was collected for subsequent measurements.Then, 100 μL of ATP detection working solution was added to each well, and the plate was incubated at room temperature for 5 min until all the background ATP was consumed, thus reducing the background.After 5 min, 20 μL of the sample or standard sample was added to detect the concentration in each well, and at least 2 s later, the relative light unit (RLU) value was measured by using a Multimode microplate reader (TECAN, Spark).

Laser scanning confocal microscopy
Briefly, SH-SY5Y cells were seeded onto glass-bottom dishes for 24 h and incubated in 2.5 μM TPA-BT-SCP dispersion at 37

MPTP-induced PD mouse model
Adult male C57BL/6J mice of approximately 25-30 g were the preferred strain, gender, and weight for MPTP-induced mouse model studies.They were randomly divided into five groups (eight mice per group).The control group was treated with saline solution (vehicle).The MPTP group was injected intraperitoneally with MPTP (dissolved in saline solution) at a final concentration of 25 mg kg −1 daily for 7 consecutive days.After the final treatment, behavioral testing, including a pole test, suspension test, swimming test, and rotarod test, was conducted to assess behavioral changes.All the evaluators were blinded to the treatment groups.

Pole test
All mouse behavioral instruments were purchased from the Zhishuduobao biotechnology company.The pole test was implemented to evaluate the movement disorder of the mice.The apparatus consisted of a wooden pole (50 cm in high, 0.5 cm in diameter, wrapped with gauze to prevent slipping) with a wooden ball at the top.The base of the pole was covered with bedding as protection for mice from injury.After acclimatization, the mice were pretrained with the pole three times to make sure that all animals would turn their head down once they were put on the ball.During the pole test, the total time it took for the mouse to get from the top to the bottom was measured.

Suspension test
This test was used to evaluate the coordination of limb movements in mice.Hang the mouse's front paws on a hemp rope placed horizontally 30 cm from the ground.After acclimatization, suspend the mouse on a horizontal wire, if the mouse grasps the wire with two hind paws, note 3 points, 2 points if they grasp the wire with one of the hind paws.If the claws cannot grasp the wire, 1 point is recorded, and the score is cal-culated.The mice received three trials, and the average time was analyzed.

Forced swimming test
This test was used to evaluate the limb coordination ability and motility of the mice.The mice were pretrained three times.After acclimatization, mice were placed in a glass tank with a water depth of 15 cm and 22-25 • C water.Each mouse was gently placed in water.The duration of immobility was recorded during the last 1 min.The scoring standards are as follows: the mouse of a continuous swimmer within 1 min is scored 3 points; the mouse of most of the time swimming only occasionally floats is scored 2.5 points; the mouse of floating time accounts for more than 50% of the entire test time is scored 2 points; mouse of occasional swimmers is scored 1.5 points; mouse of occasionally swimming with hind limbs and floating on one side is scored 1 point; those with no movement of limbs is scored 0 point.The mice received three trials, and the average time was analyzed.

Rotarod test
Mouse motor coordination was evaluated by using a rotarod apparatus.Before administration, all mice were trained on the rotarod (rotating rod diameter: 3 cm).The rotating rod device is accelerated at a constant rate of 1-25 rpm for 300 s, and the time of each test is recorded.This training process was performed for more than three rounds to train all mice to walk on the rotarod.After MPTP treatment, the rotarod test was conducted at a uniformly accelerating speed from 5 to 30 r/min in 300 s, and the latency to fall was recorded.Allow 180 s rest between each test.

in vivo TPA-BT-SCP probes imaging
The MPTP-induced mice and sham mice were first anesthetized with 3.5% chloral hydrate through intraperitoneal injection.Following shaving, the mice were fixed on a brain stereotaxic apparatus.The scalp was cut and a hole was drilled at a certain position on the skull (3.0 mm lateral, 1.3 mm posterior from bregma).A 5 μL of Hamilton syringe with a needle was then inserted through the hole into the SN at 4.7 mm below the horizontal plane of bregma.TPA-BT-SCP probes (2.5 μM, 2 μL) were infused at the SN.Then, the scalp was sewn, and animal imaging was conducted at different time points following brain stereotaxic injection of TPA-BT-SCP probe (imaging times: 0, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, and 96 h post-injection, respectively).For intranasal administration, awake mice were fixated and small drops of 10 μL TPA-BT-SCP probes (2.5 μM) were applied with a pipettor in front of the nasal cavity and inhaled by the mice.Following intranasal administration of the TPA-BT-SCP probe, animal imaging was conducted at different tome points (imaging times: 0, 0.5, 1, 4, and 24 h postadministration, respectively).The IVIS fluorescence imaging system was utilized by placing the anesthetized mouse on the equipped platform (Ex = 510 nm, Em = 670 nm).The intensity of the fluorescent signals was quantified by average radiance from a fixed-area region of interest (ROI) over the brain.

Noninvasive early diagnosis of PD in vivo
For intranasal administration, awake mice were fixated, and small drops of 10 μL TPA-BT-SCP probes (2.5 μM) were applied with a pipettor in front of the nasal cavity and inhaled by the mice.After 30 min of injection, the IVIS fluorescence imaging system was utilized by placing the anesthetized mouse on the equipped platform (Ex = 510 nm, Em = 670 nm).

Biodistribution
After intranasal administration of the TPA-BT-SCP probe to mice, they were sacrificed at different time points (0, 0.5, 1, 4, and 24 h, respectively).Major tissues were harvested and homogenized in 0.2 mL of 1% Triton X-100 using a homogenizer for 4 min.The samples were then dissolved with 0.3 mL of DMSO and incubated at room temperature overnight.After that, the solution was centrifuged at 15,000 rpm for 30 min and supernatants were transferred to a 96-well plate to determine the absorption at 510 nm.TPA-BT-SCP in the supernatant was determined by absorption based on a calibration curve and expressed as an injected dose of tissue (%ID).

Immunofluorescent staining
Briefly, the sliced tissues of mice brains on a coverslip were placed in 50 • C ovens for 30 min.Then, slices were washed with 1×TBST twice, and blocked with 0.4% Triton X-100 in BSA buffer for 1 h at room temperature.After washing with 1×TBST three times, the sections were incubated with anti-Tyrosine Hydroxylase (1:200) and anti-Tim23 (1:200) overnight at 4 • C. Subsequently, the tissue sections were washed three times with 1×TBST and incubated with the secondary antibody, fluorescein AMAD-conjugated Goat anti-Rabbit IgG (1:200), and (FITC)-conjugated Goat anti-Mouse IgG (1:200) for 2 h at room temperature in a shading box.After rinsing sections with 1×TBST three times, singlestained slides were dropped with glycerol containing DAPI, the images were captured using an upright fluorescence microscope.

Data analysis
Statistical comparisons were made by unpaired Student's ttest (between two groups) and one-way ANOVA (for multiple comparisons).p Value < 0.05 was considered statistically significant.All statistical calculations were carried out with GraphPad Prism (version 8.0.2).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

E T H I C S S TAT E M E N T
This study was performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and Guideline for Ethical Review of Animals (China, GB/T 35892-2018).All animal studies were conducted under the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University (approval ID: 2021-SYDWLL-000318).

F I G U R E 1
Characterization of TPA-BT-SCP.(A) Synthetic route of TPA-BT-SCP probe.(B) Optimized ground geometries at the highest unoccupied molecular orbital of TPA-BT-SCP (LUMO and HOMO).(C) The electronic energy levels of TPA-BT-SCP.(D) PL spectra of TPA-BT-SCP in THF/water mixture with various fw as indicated (the excitation wavelengths of TPA-BT-SCP is 670 nm).(E) Normalized absorption of TPA-BT-SCP in THF (Pure).(F) FL spectra of TPA-BT-SCP in water and DMSO mixture (Water: DMSO = 99:1).

F I G U R E 3
In vivo diagnosis of PD mice in different severities.(A-D) Behavioral testing of MPTP-induced PD mouse model.The results of (A) pole test, (B) suspension test, (C) swimming test, and (D) rotarod test of mice after 1, 3, 5, and 7 consecutive days of modeling, respectively (n = 8, each group).(E) Representative in vivo fluorescence images of saline-treated (Sham) and MPTP-induced (D1, D3, D5, and D7) PD mice after injection of the TPA-BT-SCP.(F) The corresponding average fluorescence intensity of (E) (n = 3 biologically independent mice per group).(G) Representative tissue immunofluorescence images of TH-positive cells (blue), Tim23-positive cells (green), and TPA-BT-SCP probe-positive cells (red) in the substantia nigra (SN) of Sham group and MPTP-induced group (up) and the detailed images of the subchannel (below).Scale bar: 25 μm (up) or 2.5 μm (below).Brain tissues were harvested from three mice in each group, and the representative images from each group are shown.

F I G U R E 4
in vivo noninvasive early diagnosis of PD mice.(A) Schematic illustration of intranasal instillation of TPA-BT-SCP.(B) Representative in vivo fluorescence images of saline-treated (Sham) and MPTP-induced (D1, D3, D5, and D7) PD mice taken 30 min after the instillation of TPA-BT-SCP.(C) The corresponding average fluorescence intensity of (B) (n = 3 biologically independent mice per group).Data are expressed as mean ± S.E.M. and analyzed by one-way ANOVA.*p < 0.05, **p < 0.01, ***p < 0.001.

F I G U R E 5
TPA-BT-SCP probe for early PD diagnosis in vivo.(A) Highly emission during TPA-BT-SCP probe aggregation.(B) Positively charged TPA-BT-SCP probes aggregate on mitochondrial membranes via charge-coupled interaction.(C) Schematic illustration of the in vivo diagnosis of early PD with the turn-off TPA-BT-SCP probe.
• C for 0, 2, 4, 8, and 12 h, respectively.Then, different concentrations of MPP + -induced PD SH-SY5Y cells were incubated with TPA-BT-SCP at 37 • C for 4 h.Meanwhile, mitochondrial marker Mito-tracker was used to evaluate the degree of uptake and mitochondrial localization of TPA-BT-SCP in SH-SY5Y cells with different incubation time.Cells in dishes were then washed with PBS and incubated separately for 30 min in PBS with Mitotracker.Stained cells were washed, incubated in PBS, and visualized under a laser confocal scanning microscope (TPA-BT-SCP probe, Ex = 510 nm, Em = 670 nm; MitoTracker green, Ex = 488 nm, Em = 516 nm).The fluorescence intensity and PCC were obtained from Carl Zeiss Zen image processing and analysis software.
L.H. and Y.Z.contributed equally to this work.X.X. and D.D. conceptually designed the experimental strategy.X.X.provided intellectual input and supervised the project.X.X. and Y.S. revised the paper.Y.Z., L.H., H.W., J.R., and D.J. wrote the manuscript.Y.Z., L.H., and J.R. performed the in vivo experiments.Y.Z. and L.H. performed the in vitro experiments.D.J. performed the nanomaterials preparation.Y.Z., L.H., J.R., D.J., Y.Q., D.D., and X.X.analyzed the data and participated in the discussion.All authors read the manuscript, commented on it, and approved its content.This work was supported by the National Natural Science Foundation of China (grant nos.82241058, 31922045, and 31771031), Natural Science Foundation of Tianjin Province of China (grant no.21JCZDJC00290), the State Key Laboratory of Medicinal Chemical Biology in Nankai University (grant no.2020017), and Open Funding Project of the State Key Laboratory of Biochemical Engineering (grant no.2021KF-01).