Multiplication ratio fluorescent probe for epileptic focus positioning and preparation method of multiplication ratio fluorescent probe

Abstract

The invention belongs to the technical field of molecular imaging, and particularly relates to a ratiometric fluorescent probe for epileptic focus positioning and a preparation method of the ratiometric fluorescent probe. The ratiometric fluorescent probe comprises three components with complementary functions, namely, PEG-b-PC7N which is a pH response core and is sensitive to pathological pH change, and a fluorescent probe which is sensitive to pathological pH change. A PEG-b-PC7N-b-Cy5 fluorescent pair and a PEG-b-PC7N-b-Cy7 fluorescent pair are used for responding to ROS (reactive oxygen species) and H < + > related to neuroinflammation at the focus of infection; rAP-PEG-b-PC7N is a targeting unit, and is used for promoting blood brain barrier crossing and specific epilepsy targeting. The fluorescent probe has excellent pH / ROS responsiveness, sensitivity and stability, provides high-sensitivity and user-friendly intraoperative imaging, and can significantly prolong an imaging window. The probe is expected to realize clinical transformation, can accurately visualize the epilepsy focus, and expands the crowd suitable for epilepsy surgery.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular imaging, and in particular relates to a ratiometric fluorescent probe and a preparation method thereof. Background Art

[0002] Epilepsy is a serious neurological disorder characterized by recurrent and unpredictable seizures caused by an imbalance between neuronal excitation and inhibition in the central nervous system. Currently, approximately 50 to 70 million people worldwide suffer from epilepsy. Approximately 30% of patients with epilepsy fail to respond to clinical antiepileptic drugs, resulting in refractory epilepsy, which is often associated with poor outcomes, including severe complications, injury, and even death. Frequent and severe seizures can lead to further brain damage, persistent neurobehavioral abnormalities, and neuropsychiatric disorders. Neurosurgery to remove the epileptic lesion is an effective treatment for drug-resistant epilepsy. However, the success of surgical intervention depends on a delicate balance between complete resection of the epileptic lesion and preservation of functional cortical areas. Residual epileptic tissue, often due to incomplete surgical resection, is a major cause of poor surgical outcomes in patients with drug-resistant epilepsy. For patients with frontotemporal neocortical epilepsy, surgical cure rates are suboptimal, ranging from 25% to 40%, and postoperative recurrence rates are high. Furthermore, due to the complexity and high cost of localizing the epileptic lesion, it is estimated that at least 50% of patients with drug-resistant epilepsy are suitable candidates for epilepsy surgery, yet less than 3% undergo surgical treatment.

[0003] Several techniques based on abnormal brain activity during interictal and ictal periods, including electroencephalography (EEG), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), are used in the preoperative evaluation of epilepsy surgery. All of these methods estimate the approximate location of the epileptogenic brain region but fail to provide exact boundaries. Furthermore, the utility of these non-invasive techniques is compromised by brain shift and deformation caused by cerebrospinal fluid loss during surgery. Although electrophysiological recording methods or EEG are currently the "gold standard" for epilepsy mapping, they are insufficient to define the boundaries of the epileptic focus due to sampling limitations and volume conduction. Intracranial EEG (iEEG) can improve spatial localization accuracy, but its high invasiveness and limited recording coverage can lead to misleading localization information if it is not correctly placed over the epileptic focus. Therefore, new intraoperative methods with high temporal and spatial resolution are urgently needed to accurately localize the epileptic focus.

[0004] Over the past few decades, several optical techniques have been developed for mapping epileptic foci, offering high spatial resolution and sensitivity to neural activity. Techniques such as intrinsic optical signals (IOS) or near-infrared spectroscopy (NIRS) have been used to provide excellent surface maps of epileptic foci by imaging changes in hemoglobin oxygen saturation and local blood volume associated with epileptic activity. However, these techniques are limited by relatively small signal amplitudes (ranging from 0.1 to 5%), resulting in low imaging sensitivity. Voltage-sensitive dyes can monitor changes in membrane potential across a large number of neurons, potentially enhancing imaging sensitivity. However, these dyes have difficulty crossing the blood-brain barrier (BBB) and typically require topical application. Prolonged perfusion procedures can increase the risk of intraoperative anesthetic exposure and the likelihood of missing deep lesions due to the dye's low permeability. The increased neuronal excitability caused by epilepsy also enhances anaerobic glycolysis, leading to lactate accumulation and a significant decrease in pH, which can be used for imaging. However, responding to pH alone lacks specificity for epileptic foci. Recurrent epileptic seizures also induce neuroinflammation, increasing myeloperoxidase (MPO) expression locally at the epileptic site. Summary of the Invention

[0005] The purpose of the present invention is to provide a multiplicative ratio fluorescent probe for epileptic focus localization that can respond to multiple targets simultaneously and has high spatiotemporal resolution and a preparation method thereof, which is used for highly sensitive and user-friendly intraoperative imaging to accurately locate the epileptic focus (EF), thereby improving the success rate of epilepsy surgery.

[0006] The multiplication ratio fluorescent probe for epileptic focus localization provided by the present invention integrates three components with complementary functions, and the general structural formula is as follows:

[0007]

[0008] in:

[0009]

[0010] The three components are:

[0011] (1) PEG-b-PC7N, a pH-responsive core, responds sensitively to pathological pH changes;

[0012] (2) PEG-b-PC7N-b-Cy5 and PEG-b-PC7N-b-Cy7, a FRET-based fluorescence pair, respond to ROS and H associated with neuroinflammation in lesions. + ;

[0013] (3) RAP-PEG-b-PC7N is a targeting unit that promotes blood-brain barrier crossing and specific epilepsy targeting.

[0014] Explanation of symbols: PEG is polyethylene glycol; b represents the connector symbol in the block copolymer, e.g., PEG-b-PC7N represents an amphiphilic copolymer formed by covalently linking polyethylene glycol (PEG) blocks and PC7N blocks; PC7N represents a hydrophobic segment formed by polymerization of the monomer C7N, C7N monomer structure: 2-(hexamethyleneimino)ethyl methacrylate (a nitrogen-containing heterocyclic structure); Cy5 and Cy7 represent anthocyanin fluorescent dyes, where Cy5 emits red fluorescence at approximately 670 nm and serves as a fluorescence donor; Cy7 emits near-infrared fluorescence at approximately 800 nm and serves as a fluorescence acceptor. CTA is a chain transfer agent, and Mal is maleimide.

[0015] The present invention also provides a method for preparing the multiplication ratio fluorescent probe, which comprises the following steps:

[0016] (1) Preparation of PEG-b-PC7N;

[0017] PEG-b-PC7N was prepared by adding PEG-CTA, 2-(azepan-1-yl)ethyl methacrylate, and AIBN at a molar ratio of PEG-CTA:2-(azepan-1-yl)ethyl methacrylate:AIBN=(5-8):(50-60):1 to a glass ampoule containing 1 mL of 1,4-dioxane and 1 mL of DMSO, degassing, sealing, and heating in an oil bath at 85-100°C to obtain PEG-b-PC7N.

[0018] (2) Preparation of PEG-b-PC7N-b-Cy5 and PEG-b-PC7N-b-Cy7 fluorescent pairs;

[0019] Synthesis of PEG-b-PC7N-b-Cy7:

[0020] First, ACy7S was prepared by reacting K2CO3, allyl bromide, and compound 9: 3H-indole cation, 2-[2-[2-[(2-carboxyethyl)thio]-3-[2-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]vinyl]-1,3,3-trimethyl in acetone at a molar ratio of K2CO3:allyl bromide:compound 9 = (1.1-1.3):(1.2-1.5):1 to obtain ACy7S;

[0021] PEG-b-PC7N, ACy7S and AIBN were added to a glass ampoule containing 0.3-0.5 mL of 1,4-dioxane and 0.3-0.5 mL (preferably 0.3 mL) of DMSO at a molar ratio of PEG-b-PC7N:ACy7S:AIBN=2:(7-10):(0.0056-1) to react by reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare PEG-b-PC7N-b-Cy7;

[0022] Synthesis of PEG-b-PC7N-b-Cy5:

[0023] Compound 3 (DR1-6) and triethylamine (TEA) are dissolved in 20-25 ml of anhydrous dichloromethane (DCM) at a molar ratio of 1-2:3.585 (preferably 1:3.585), and stirred at -1-0°C (preferably 0°C) for 30-35 (preferably 30) minutes under nitrogen protection; acryloyl chloride is then added dropwise at a molar ratio of compound 3:acryloyl chloride = 1:2-2.5 (preferably 2); the mixture is warmed to room temperature and stirred overnight; the solvent is then removed under reduced pressure, and the residue is purified by silica gel column chromatography to obtain ACy5 as a dark blue powder;

[0024] PEG-b-PC7N, ACy5, and AIBN were added to a glass ampoule containing 0.3-0.5 mL of 1,4-dioxane at a molar ratio of PEG-b-PC7N:R-Cy5:AIBN=2:(7-10):(0.0056-0.007) and reacted using reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare PEG-b-PC7N-b-Cy5.

[0025] (3) Preparation of RAP-PEG-b-PC7N;

[0026] CPADB-NHS, NH2-PEG 113 -Mal with CPADB-NHS:NH2-PEG 113 -Mal=(0.05~0.07)mmol:(0.06~0.08)mmol) was added to a round-bottom flask containing 3~5mL anhydrous CH2Cl2 to react and prepare CTA-PEG 113 -Mal;

[0027] CTA-PEG-Mal, 2-(azepan-1-yl)ethyl methacrylate, and AIBN were added to a glass ampoule containing 1-3 mL of 1,4-dioxane at a ratio of CTA-PEG-Mal:2-(azepan-1-yl)ethyl methacrylate:AIBN = (0.004-0.006 mmol):(0.4-0.6 mmol):(0.0008-0.0010 mmol) to carry out RAFT polymerization to prepare PEG-b-PC7N-Mal.

[0028] PEG-b-PC7N-Mal and RAP peptide were dissolved in PBS (pH 6.4-6.6) at a mass ratio of PEG-b-PC7N-Mal:RAP peptide = (20-25 mg): (4-6 mg) (preferably 20:4 mg), and the two solutions were mixed, and N2 was bubbled through the mixture, and the reaction was carried out at room temperature for 12-15 hours (preferably 12 hours) to prepare RAP-PEG-b-PC7N;

[0029] (4) Finally, a pH-responsive type with targeted function was synthesized The method comprises the following steps: dissolving 1 to 2 mg of a pH-responsive polymer fluorescent donor PEG-b-PC7N-Cy5, 5 to 6 mg of a pH-responsive polymer fluorescent acceptor PEG-b-PC7N-Cy7, 0.05 to 0.10 mg of a RAP peptide-modified pH-responsive polymer RAP-PEG-b-PC7N, and 0.05 to 0.10 mg of a pH-responsive core PEG-b-PC7N in 1 to 2 mL of methanol; then, ultrasonically treating the mixture for 1 to 2 minutes; then, rapidly adding 9 to 12 mL of deionized water to the mixture, and continuing ultrasonic treatment for 10 to 15 minutes; evaporating the methanol at 40 to 50° C.; and washing the resulting nanoparticles with deionized water using a 100 kDa centrifugal filter tube and resuspending them three times, and then storing them at 4 to 5° C.

[0030] In the multiplication ratio fluorescent probe provided by the present invention, the three components contain 4 micelles (see Figure 1 、 Figure 5 The fluorescent probe composed of the micelle system has excellent pH / ROS responsiveness, sensitivity and stability, providing highly sensitive and user-friendly intraoperative imaging and significantly extending the imaging window. The probe starts with the self-assembly of functional monomers into micelles, generating a molecule between Cy5 and Cy7. The resonance energy transfer (FRET) effect results in a low Cy5 / Cy7 ratio. After intravenous injection, the probe crosses the blood-brain barrier via RAGE-mediated transcellular transport. In the acidic microenvironment of the epileptic lesion, protonation of the polymer triggers micelle disruption, reducing the FRET effect and increasing the Cy5 / Cy7 ratio. Subsequently, the oxidative conditions of the epileptic lesion degrade Cy7, further increasing the Cy5 / Cy7 ratio. The high Cy5 / Cy7 ratio exhibits specific fluorescence characteristics, enabling imaging; in addition, the pH-sensitive dye-coupled cationic polymer induced by the acidic microenvironment is easily internalized and locally retained by brain cells, thereby extending the imaging window for convenient surgical operations. Due to its high sensitivity and biocompatibility, this probe has the potential for clinical translation, enabling precise visualization of epileptic lesions and expanding the population suitable for epilepsy surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The screening, synthesis, and principles of polymers are demonstrated. Figure a shows the degree of proton transfer for different polymers at different protonation levels. Figure b is used to screen and optimize the initial ratio of donor and acceptor. Figure c is used to optimize the target head ratio to improve cross-BBB properties. Figure d shows the polymer structure and mechanism of action, illustrating how the polymer localizes within the cell and affects the intracellular environment through ROS (reactive oxygen species) and H+. Figure de shows the secondary amplification effect of the ratio after the secondary cascade response of the probe in the lesion.

[0032] Figure 2 In vitro FRET (fluorescence resonance energy transfer) fluorescence effect evaluation is demonstrated. Ab indicates that charge reversal after polymer response enhances intracellular retention, and de is a cell model used to verify probe effectiveness. PBS and LPS induce ROS, while KA induces a pH decrease and ROS upregulation. Fus inhibits pH decrease after KA induction, and Lor is used to inhibit ROS upregulation after KA induction.

[0033] Figure 3 Fluorescence detection of epileptic foci in a mouse model. Figure a is a visual experimental flow chart illustrating the experimental process, including KA model establishment, probe injection, ECOG acquisition, ratiometric fluorescence imaging, and histopathological analysis. Figure b shows actual images of the PBS group after injection of the targeted-responsive probe, the KA group after injection of the non-targeted-responsive probe, the KA group after injection of the targeted-unresponsive probe, and the KA group after injection of the targeted-responsive probe, demonstrating the in vivo imaging effect of the polymer. Figures c and d show the effects of the probe in the electric kindling model and the pilocarpine model, demonstrating the universal applicability of the probe in epilepsy models.

[0034] Figure 4This probe is used to image epileptic foci in a mouse model and verify surgical outcomes. (a) shows the steps involved in resecting a residual lesion following ECoG-guided surgical resection. (b) shows images of the residual lesion after ECoG-guided surgical resection using a ratiometric fluorescence imaging probe. (c) and (d) show spectral and ROI analysis, demonstrating that the resected tissue ex vivo confirms that it is anomalously firing electrical tissue.

[0035] Figure 5 This is a diagram illustrating the composition of the multiplication ratio fluorescent probe used for localizing epileptic foci according to the present invention.

[0036] Figure 6 For electroencephalogram. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples with reference to the accompanying drawings, but the present invention is not limited to the scope of the examples. Experimental methods without specific conditions specified in the examples were performed according to conventional methods and conditions, or selected according to the product specifications.

[0038] Example 1: Synthesis of a multiplication ratio fluorescent probe; the specific steps are:

[0039] (I) Synthesis of pH-sensitive groups (C5N(2-(tetramethyleneimino)ethyl methacrylate), C6N(2-(pentamethyleneimino)ethyl methacrylate), and C7N(2-(hexamethyleneimino)ethyl methacrylate)).

[0040]

[0041] (i) Hydroquinone, triethylamine, tetrahydrofuran, 70°C, 2 hours.

[0042] The synthesis of C5N, C6N, and C7N was based on a previously published literature using similar procedures. (Zhou, K., Wang, Y., Huang, X., Luby-Phelps, K., Sumer, BD, & Gao, J. (2011). Tunable, ultrasensitive pH-responsive nanoparticles targeting specific endocytic organelles in living cells. Angewandte Chemie International Edition , 50(27),6109-6114.)

[0043] Synthesis of C5N (2-(tetramethyleneimino)ethyl methacrylate): First, N-(2-hydroxyethyl)pyrrolidine (11.5 g, 0.1 mol), triethylamine (TEA, 10.1 g, 0.1 mol) and the inhibitor hydroquinone (0.11 g, 0.001 mol) were dissolved in 100 ml of tetrahydrofuran (THF), and then methacryloyl chloride (10.4 g, 0.1 mol) was added dropwise to a three-necked flask. The solution was refluxed in THF for 2 hours. After the reaction was completed, the precipitated triethylamine hydrochloride was removed by filtration, and the THF solvent was removed by rotovap. The final residue was distilled in vacuum (83-87°C, 0.05 mmHg) to obtain a colorless liquid.

[0044] Synthesis of C6N (2-(Pentamethyleneimino)ethyl methacrylate): 2-(Pentamethyleneimino)ethanol (12.9 g, 0.1 mol), triethylamine (10.1 g, 0.1 mol), and the polymerization inhibitor hydroquinone (0.11 g, 0.001 mol) were dissolved in 100 mL of tetrahydrofuran. Methacryloyl chloride (10.4 g, 0.1 mol) was then added dropwise to a three-necked flask. The solution was refluxed in tetrahydrofuran for 2 hours. After the reaction was complete, the precipitated triethylamine hydrochloride was removed by filtration, and the tetrahydrofuran solvent was then removed by rotary evaporation. The resulting residue was distilled under vacuum (83-87°C, 0.05 mmHg) to obtain a colorless liquid.

[0045] Synthesis of C7N (2-(hexamethyleneimino)ethyl methacrylate): 2-(Hexamethyleneimino)ethanol (22.54 g, 0.1 mol), triethylamine (10.1 g, 0.1 mol), and the polymerization inhibitor hydroquinone (0.11 g, 0.001 mol) were dissolved in 100 mL of tetrahydrofuran. Methacryloyl chloride (10.4 g, 0.1 mol) was then added dropwise to a three-necked flask. The solution was refluxed in tetrahydrofuran for 2 hours. After the reaction was complete, the precipitated triethylamine hydrochloride was removed by filtration, and the tetrahydrofuran solvent was then removed by rotary evaporation. The resulting residue was distilled under vacuum (83-87°C, 0.05 mmHg) to obtain a colorless liquid.

[0046] (II): Synthesis of ACy5. The synthetic route is:

[0047]

[0048] condition:

[0049] (ii) TEA, ethanol, 80°C, 12 h;

[0050] (iii) TEA, dichloromethane, nitrogen, acryloyl chloride, 25°C, 12 hours.

[0051] Synthesis of ACy5:

[0052] Compound 1 (N,N,N-trimethyl-N-[1-(N-ethyl-N-methoxy)phenyl]-1,3-butadiene quaternary ammonium salt), compound 2 (1-(2-carboxyethyl)-2,3,3-trimethyl-3H-indol-1-ium) and compound 3 (DR1-6) were synthesized according to the published literature (Wang, Y., Lei, Z., Wang, C., Cao, C., Hu, J., Du, L.,...&Li, C. (2021). Unsymmetrical pentamethine cyanines for visualizing physiological acidities from the whole-animal to the cellular scale with pH-responsive deep-red fluorescence. RSCadvances, 11(29), 17871-17879.).

[0053] Compound 3 (85.4 mg, 0.2 mmol) and triethylamine (TEA, 0.1 mL) were dissolved in anhydrous dichloromethane (DCM, 20 mL) and stirred at 0°C for 30 minutes under nitrogen. Acryloyl chloride (362 mg, 0.4 mmol) was then added dropwise. The mixture was warmed to room temperature and stirred overnight. The solvent was then removed under reduced pressure, and the residue was purified by silica gel column chromatography (DCM: methanol = 90:10) to obtain ACy5 as a dark blue powder (54 mg, 56% yield).

[0054] H NMR (1H NMR, 400 MHz, CDCl3) δ: 8.23–8.10 (m, 2H), 7.47–7.35 (m, 6H), 7.15 (t, J = 8.4 Hz, 2H), 6.98 (t, J = 12.5 Hz, 1H), 6.56 (d, J = 13.4 Hz, 1H), 6.48 (d, J = 13.6 Hz, 1H), 6.31 (dd, J1 = 17.3, J2 = 1.4 Hz, 1H) ,6.00(dd,J1=17.3,J2=10.5Hz,1H),5.82(dd,J1=10.4,J2=1.4Hz,1H),4.68(t,J=5.4Hz,2H), 4.53(t,J=5.5Hz,2H), 4.22(t,J=7.5Hz,2H), 1.80(s,6H), 1.78(s,6H), 1.49(t,J=7.1Hz,3H).

[0055] Electrospray ionization mass spectrometry (ESI-MS) calculated value: C 32 H 37 N2O2 + [M]+: 481.2850, measured value: 481.2854.

[0056] (III): Synthesis of ACy7 S. The synthetic route is:

[0057]

[0058] Condition (iv): K2CO3, allyl bromide, acetone, 12h, 60℃;

[0059] Synthesis of Acy7 S:

[0060] K2CO3 (13 mg, 0.11 mmol), allyl bromide (145 mg, 0.12 mmol) and compound 4 (3H-indolyl cation, 2-[2-[2-[(2-carboxyethyl)thio]-3-[2-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]vinyl]-1,3,3-trimethyl) (58.1 mg, 0.1 mmol) were mixed in 5 mL of acetone and refluxed overnight. After evaporation of the solvent, the crude product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 99:1) to obtain ACy7S as a blue powder (40 mg, 65% yield).

[0061] NMR data:

[0062] 1 H NMR (400MHz, CDCl3): δ: 8.75 (d, J = 14.1 Hz, 2H), 7.44–7.34 (m, 4H), 7.23 (d, J = 7.4, 2H), 7.16(d,J=7.9Hz,2H),6.26(d,J=14.1Hz,2H),5.93–5.80(m,1H),5.33–5.19(m,2H),4.5 6(dd,J1=5.8,J2=1.5Hz,2H),4.24(q,J=7.2Hz,4H),3.05(t,J=7.2Hz,2H),2.69(t,J=6. 2Hz,4H),2.64(t,J=7.2Hz,2H),1.97–1.91(m,2H),1.72(s,12H),1.46(t,J=7.2Hz,6H).

[0063] 13C NMR (151MHz, CDCl3): δ: 171.59, 170.91, 156.21, 145.60, 141.93, 141.03, 134.27 ,131.75,128.84,125.11,122.25,118.77,110.55,101.14,74.28,65.61,49.20,3 9.81, 34.98, 32.31, 31.93, 31.64, 31.44, 30.20, 29.70, 29.66, 29.48, 29.36, 29.31, 29.12, 27.95, 26.61, 26.59, 24.86, 22.76, 22.69, 20.80, 17.70, 14.12, 12.42.

[0064] Mass spectral data: ESI-MS calculated value is C 40 H 49 N2O2S + [M] + : 621.3509, the measured value is 621.3515.

[0065] (IV): pH sensitive polymer (PEG 113 -b-PCXN) synthesis, the synthetic route is:

[0066]

[0067] (1) Synthesis of PEG-b-PCXN polymer.

[0068] The conditions for each stage are:

[0069] (v) N-hydroxysuccinimide, dicyclohexylcarbodiimide (DCC), dichloromethane (DCM), 20 °C, 18 h;

[0070] (vi) polyethylene glycol-amine (mPEG-NH2), dichloromethane (DCM), 37 °C, 20 h;

[0071] (vii) Azobisisobutyronitrile (AIBN), cyano compound (CXN), 1,4-dioxane, dimethyl sulfoxide (DMSO), 85°C, 12h.

[0072] A method for synthesizing a polyethylene glycol (PEG) macromolecular chain transfer agent (CTA-mPEG) is described in the literature (Warren, NJ, Mykhaylyk, O.O., Mahmood, D., Ryan, AJ, & Armes, SP (2014). RAFT aqueous dispersion polymerization yields poly(ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies. Journal of the American Chemical Society, 136(3), 1023-1033.). The synthesis of pH-sensitive polymer PEG-b-PCXN and pH-insensitive polymer PEG-b-PEH adopted the previously reported reversible addition-fragmentation chain transfer (RAFT) method (Wang, K., Jiang, M., Li, T., Liu, Y., Zong, Q., Xu, Q., ... & Yuan, Y. (2024). A Synergistic Chemoimmunotherapy System Leveraging PD-L1 Blocking and Bioorthogonal Prodrug Activation. Advanced Materials, 36(30), 2402322.).

[0073] Synthesis of PEG-b-PC7N: CTA-mPEG (200 mg, 0.04 mmol), 2-azaheptaneethyl methacrylate (C7N) (84.4 mg, 0.4 mmol), and azobisisobutyronitrile (AIBN) (1.312 mg, 0.008 mmol) were added to a glass ampoule containing 1 mL of 1,4-dioxane and 1 mL of dimethyl sulfoxide. The ampoule was then degassed by three freeze-thaw cycles and sealed under vacuum. Subsequently, the ampoule was immersed in an oil bath heated to 85°C and the polymerization reaction was initiated under magnetic stirring. After 12 hours, the ampoule was quenched with liquid nitrogen to terminate the polymerization reaction and precipitated in excess diethyl ether to obtain a pale yellow polymer.

[0074] Synthesis of PEG-b-PC5N33: CTA-mPEG (200 mg, 0.04 mmol), 2-(tetramethyleneimino)ethyl methacrylate (C5N) (78.9 mg, 0.4 mmol), and azobisisobutyronitrile (AIBN) (1.312 mg, 0.008 mmol) were added to a glass ampoule containing 1 mL of 1,4-dioxane and 1 mL of dimethyl sulfoxide. The ampoule was then degassed via three freeze-thaw cycles and sealed under vacuum. Subsequently, the ampoule was immersed in an oil bath heated to 85°C and the polymerization reaction was initiated under magnetic stirring. After 12 hours, the ampoule was quenched with liquid nitrogen to terminate the polymerization reaction and precipitated in excess diethyl ether to yield a yellow polymer.

[0075] pass 1 A series of mPEG-b-PCXN copolymers were confirmed by H NMR.

[0076] mPEG-b-PC5N 1 H NMR (400 MHz, CDCl 3 ) data are as follows: δ: 4.07 (s, 52H), 3.63 (m, 450H), 3.37 (s, 3H), 2.74-2.51 (m, 153H), 2.09 (s, 52H), 1.79 (s, 102H), 0.93 (m, 104H);

[0077] mPEG-b-PC6N 1 H NMR (400 MHz, CDCl 3 ) data are as follows: δ: 4.05 (s, 50H), 3.63 (m, 450H), 3.36 (s, 3H), 2.57 (m, 50H), 2.43 (s, 100H), 1.57 (s, 100H), 1.43 (s, 50H), 0.94 (m, 150H);

[0078] mPEG-b-PC7N 1 H NMR (400 MHz, CDCl 3 ) data are as follows: δ: 4.02 (s, 45H), 3.63 (m, 450H), 3.37 (s, 3H), 2.88-2.59 (m, 138H), 1.60 (m, 184H), 0.95 (m, 92H);

[0079] mPEG-b-PEH 1 H NMR (400 MHz, CDCl 3 ) data are as follows: δ: 3.99-3.70 (m, 46H), 3.64 (m, 450H), 3.37 (s, 3H), 1.79 (s, 70H), 1.60-1.15 (m, 156H), 0.89 (m, 69H).

[0080] (V): Synthesis of the targeting group RAP-PEG-b-PC7N copolymer. The synthetic route is:

[0081]

[0082] The conditions for each stage are:

[0083] (viii) NH2-PEG-Mal, dichloromethane (DCM), 37°C, 20 h;

[0084] (ix) C7N, azobisisobutyronitrile (AIBN), 1,4-dioxane, 80 °C, 16 h;

[0085] (x) RAP, triethylamine (TEA), acetone, 37°C, 12h.

[0086] (1) CTA-PEG 113 -Synthesis of Mal.

[0087] CPADB-NHS was synthesized as reported (Warren, NJ, Mykhaylyk, OO, Mahmood, D., Ryan, AJ, & Armes, SP (2014). RAFT aqueous dispersion polymerization yields poly(ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies. Journal of the American Chemical Society, 136(3), 1023-1033.). Before the reaction, all glassware was dried at 120°C for 2 hours to remove moisture. Then, CPADB-NHS (18.85 mg, 0.05 mmol) was dissolved in 3 mL of anhydrous CH2Cl2 and added to a round-bottom flask. Nitrogen was bubbled through the flask until the air was displaced. Next, NH2-PEG 113 -Mal (300 mg, 0.06 mmol) was dissolved in 3 mL of anhydrous dichloromethane and added dropwise to the flask. The mixture was stirred at room temperature overnight. After the reaction was complete, the product was precipitated with excess cold ether.

[0088] 1H NMR (400MHz, CDCl3) δ: 7.91 (d, J=7.8Hz, 1H), 7.60-7.49 (m, 1H), 7.39 (m, 2H), 6.7 0(s, 2H), 3.64(s, 411H), 3.41-3.20(m, 6H), 2.58-2.47(m, 4H), 1.28-1.15(m, 7H)

[0089] (2) Synthesis of PEG-b-PC7N-Mal.

[0090] PEG-b-PC7N-Mal was prepared using RAFT polymerization. Typically, CTA-PEG-Mal (20 mg, 0.004 mmol), compound 3 (84.4 mg, 0.4 mmol), and AIBN (0.14 mg, 0.0008 mmol) were added to a glass ampoule containing 1 mL of 1,4-dioxane.

[0091] 1 H NMR (400MHz, CDCl3) δ: 6.53 (m, 2H), 4.02 (s, 99H), 3.63 (m, 450H), 3.40 (m, 3H), 2.82-2.65 (m, 300H), 1.71-1.55 (m, 421H), 1.44-0.64 (m, 200H).

[0092] (3) Synthesis of RAP-PEG-b-PC7N.

[0093] 20 mg of PEG-b-PC7N-Mal was dissolved in 2 mL of PBS (pH 6.4), and 4 mg of RAP peptide was dissolved in 4 mL of PBS. The two solutions were then mixed and N2 was bubbled through the mixture. The reaction was carried out at room temperature for 12 hours. The mixture was then dialyzed in deionized water for 24 hours using an ultrafiltration tube (3500 Da), and the resulting solution was lyophilized to obtain RAP-PEG. 113 -b-PC7N.

[0094] 1 H NMR (400MHz, CDCl3) δ: 4.25 (m, 99H), 3.53 (m, 450H), 2.81-2.51 (m, 300H), 2.35 (m, 100H), 2.08-1.72 (m, 208H), 1.59 (m, 303H), 0.9-0.82 (m, 202H).

[0095] (VI): Synthesis of PEG-b-PC7N-Cy5 / PEG-b-PC7N-Cy7 Copolymers Based on FRET Fluorescence

[0096]

[0097] Synthetic conditions:

[0098] (xi) Acy5, azobisisobutyronitrile (AIBN), dimethyl sulfoxide (DMSO), 1,4-dioxane, 80 °C, 12 h;

[0099] (xii) Acy7S, azobisisobutyronitrile (AIBN), dimethyl sulfoxide (DMSO), 1,4-dioxane, 80°C, 12h.

[0100] (1) Synthesis of PEG-b-PC7N-b-dye.

[0101] Preparation of PEG using RAFT polymerization technology 113 -b-PC7N-b-Cy5 and PEG 113 -b-PC7N-b-Cy7.

[0102] PEG 113 Synthesis of -b-PC7N-b-Cy7:

[0103] PEG 113 -b-PC7N (20 mg, 0.014 mmol), R-Cy7 (4.47 mg, 0.007 mmol) and AIBN (0.05 mg, 0.00028 mmol) were added to a glass ampoule containing 0.3 mL of 1,4-dioxane and 0.3 mL of dichloromethane and prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization technique.

[0104] PEG 113 Synthesis of β-PC7N-β-Cy5: PEG 113 -b-PC5N (27.6 mg, 0.014 mmol), R-Cy5 (5.41 mg, 0.007 mmol) and AIBN (0.05 mg, 0.00028 mmol) were added to a glass ampoule containing 0.3 mL of 1,4-dioxane and 0.3 mL of dichloromethane and prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization technique.

[0105] (VII): Synthesis of TRFM

[0106] In order to synthesize pH-responsive Resonance energy transfer (FRET) nanomicelles (TRFM), 1 mg of pH-responsive polymer fluorescent donor (PEG-b-PC7N-Cy5), 5 mg of pH-responsive polymer fluorescent acceptor (PEG-b-PC7N-Cy7), 0.05 mg of RAP peptide-modified pH-responsive polymer (RAP-PEG-b-PC7N), and 0.05 mg of pH-responsive core PEG-b-PC7N were dissolved in 1 mL of methanol. Subsequently, the mixture was sonicated for 1 minute. Next, 9 mL of deionized water was quickly added to the mixture, and sonication was continued for 10 minutes. Methanol was evaporated at 40°C. The resulting nanoparticles were washed and resuspended three times with deionized water using a 100 kDa centrifugal filter tube and then stored at 4°C.

[0107] Preparation of pH-insensitive micelles (TFM): pH-insensitive monomer (mPEG-b-PEH), 1 mg of Acy5 and 5 mg of Acy7S were used to prepare TFM by the same method as above.

[0108] Preparation of pH-responsive micelles (RFMs) without targeting function: pH-responsive monomer (mPEG-b-PC7N), 0.1 mg of RAP-PEG-b-PC7N, 1 mg of Acy5 and 5 mg of Acy7S were used to prepare the RFMs by the same method as above.

[0109] Example 2: Confocal microscopy imaging.

[0110] HT22 and SH-SY5Y cells were seeded onto glass-bottomed culture dishes suitable for confocal microscopy and incubated at 37°C, 5% CO2 for 24 hours. After 24 hours of incubation of HT22 cells with KA and LPS, different types of probes (TRFM, TFM, RFM) were added. All images were acquired using a laser scanning confocal microscope (Carl Zeiss LSM980). A 40x air objective and a 63x oil immersion objective were used. Cy5 (excitation light / excitation light: 600nm / 660nm); Cy7 (excitation light / excitation light: 600nm / 820nm). See Figure 2 Figures a and b illustrate the enhanced intracellular retention of the polymer after charge reversal, while d and e are cell models used to verify the effectiveness of the probe. PBS and LPS induce ROS, while KA induces a decrease in pH and an upregulation of ROS. Fus inhibits the decrease in pH after KA induction, and Lor is used to inhibit the upregulation of ROS after KA induction.

[0111] Example 3: Biodistribution studies and brain fluorescence imaging.

[0112] To investigate the blood-brain barrier (BBB) penetration of RAP-modified TRFM micelles, donor and acceptor NIR fluorescent molecules in the FRET micelles were used as fluorescent labels for in vivo and ex vivo assays. Male C57BL / 6 mice were randomly divided into two groups and treated differently (n = 3): (I) saline-induced TRFM-injected mice and (II) KA-induced TRFM-injected mice. Both groups were injected with the same concentration of FRET micelles (polymer monomer concentration: 5 mg / kg). In vivo fluorescence imaging was performed using an IVIS Spectrum system at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after intravenous probe injection. Each group of mice was then perfused with saline, and the corresponding brains were harvested. Brain regions were identified according to the Allen Brain Atlas: CPu: caudate putamen (striatum), NAc: nucleus accumbens, dHip: dorsal hippocampus, MD: middorsal thalamic nucleus, vHip: ventral hippocampus, SN: substantia nigra, and VTA: ventral tegmental area.

[0113] High-resolution in vivo fluorescence imaging. To obtain fluorescent signals from changes in the microenvironment of the epileptogenic focus, a series of probes were selected for more detailed imaging. Male C57BL / 6 mice were randomly divided into four groups and received different treatments (n=3):

[0114] (I) Saline-induced epilepsy sham-operated mice injected with TRFM, (II) KA-induced epilepsy model mice injected with RFM, (III) KA-induced epilepsy model mice injected with TFM, and (IV) KA-induced epilepsy model mice injected with TRFM. Both groups of mice were injected with the same concentration of FRET micelles (polymer monomer concentration: 5 mg / kg). An in vivo fluorescence imaging system (Xenogen, USA) was used to image 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours after intravenous injection of the probe. Four hours after the tail vein injection of the TRFM probe, mice in each group were killed, their brain tissues were collected and fixed, and changes in the microenvironment of the epileptogenic focus were evaluated by probe tracking. For tissue distribution studies, mice were killed, and brain tumors and major organs were collected for in vitro imaging. To obtain fluorescent images of the brain, the rat model brain was exposed to 600 nm excitation light for 1000 ms, and the Cy5 signal (filters: 620 nm sp and 720 nm lp) and the Cy7 signal (filter: 750 nm lp) were collected simultaneously. The ratio of the two fluorescent images was converted pixel by pixel into a ratiometric map. Figure 3Figure 1 shows a flow chart of the experimental procedure, including KA model establishment, probe injection, ECOG acquisition, ratiometric fluorescence imaging, and histopathological analysis. Figure 1 shows images of the PBS group after injection of the targeted-responsive probe, the KA group after injection of the non-targeted-responsive probe, the KA group after injection of the targeted-unresponsive probe, and the KA group after injection of the targeted-responsive probe, demonstrating the in vivo imaging effect of the polymer. Figures 1 and 2 show the effects of the probe in the electric kindling model and the pilocarpine model, demonstrating the universal applicability of the probe in epilepsy models.

[0115] Fluorescence Imaging-Guided Epilepsy Resection. A mouse model of epilepsy was prepared as described above and randomly divided into two groups. One group of mice received an intravenous injection of TRFM (200 nmol / kg) via the tail vein. Six to eight hours later, the mice were anesthetized with 1-2% isoflurane, and a craniotomy was performed to expose the cortical surface for in vivo fluorescence microscopy. Based on I660 / I820 intensity calculations and guided by real-time FL-Ratio maps, the epileptogenic focus was carefully resected until no tissue with a high I660 / I820 ratio could be detected. To mimic clinical conditions, another group of mice underwent surgery guided by ECoG and the expertise of the surgeon. In Vivo Fluorescence Imaging and Microscopy Studies. Mouse brains were harvested and prepared into frozen sections. Cy5 and Cy7 signals, as well as brightfield images, were captured using confocal microscopy to verify probe entry into the brain and changes in signal response. Horizontal sections of the mouse brains were then immunostained to verify colocalization of TRFM with the epileptiform lesion in vivo. Nissl staining was used to detect neurons in the mouse brains. Immunofluorescence staining was also performed on frozen tissue sections of epilepsy patient samples to verify the expression and colocalization of RAGE receptors on blood vessels. Antibody information is as follows: DAPI (Servicebio, G1012), RAGE (R&D Systems, AF1179, 1:200). CD31 (Abcam, ab24590, 1:100). See Figure 4 shown. Figure 4 This probe is used to image epileptic foci in a mouse model and verify surgical outcomes. (a) shows the steps involved in resecting a residual lesion following ECoG-guided surgical resection. (b) shows an image of the residual lesion after ECoG-guided surgical resection, detected using a ratiometric fluorescence imaging probe. (c) and (d) show spectral and ROI analyses, demonstrating that the resected tissue ex vivo confirms that it is anomalously firing electrical tissue.

[0116] Example 5: Electroencephalogram.

[0117] The specific steps are as follows: After KA (kainic acid) injection, a steel screw was manually implanted into the epidural space at the KA injection site to record the electroencephalogram (EEG). The reference electrode was placed above the cerebellum. Freely moving mice were connected to the NeuroLog electrophysiological recording system (Digitimer Ltd) using a long flexible cable, enabling monitoring of intracranial EEG activity. The recorded signals were amplified 1,000 times, filtered from 0.1Hz to 1,000Hz, digitized at a sampling rate of 2,500Hz, and recorded using Spike2 software. Cortical ECoG study. ECoG recordings were performed using the g.Recorder system (g.tec Medical Engineering GmbH, Austria). A 64-channel electrode array was created for ECoG monitoring, with electrodes evenly distributed over an area of 1.1×0.7cm2. To record ECoG, mice were anesthetized with 1-2% isoflurane, and a craniotomy was performed using a stereotaxic frame to expose the cortical surface. A custom electrode array was directly connected to the exposed cortex for real-time ECoG monitoring, and a piece of saline-soaked gelatin sponge was placed on the array to improve signal recording. Raw data were sampled at 4.8 kHz and downsampled to 1 kHz before being converted to MAT format. Preprocessing and data analysis were performed using custom MATLAB (R2013b) scripts based on the Fieldtrip Toolbox. Each channel of the raw ECoG data was re-referenced to the adjacent channel on the same electrode axis to calculate bipolar re-referencing. The re-referenced data were then notch filtered to remove 50 Hz power line noise and its harmonics and then band-pass filtered from 1 to 30 Hz. The dynamics of brain activity within the 10 seconds before the seizure was examined with a temporal resolution of 0.5 seconds. Specifically, the average ECoG signal of all channels within the window was normalized to a range of 0 to 1, where the channel with the largest amplitude was labeled 1 and the channel with the smallest amplitude was labeled 0. The peak time of a given channel was defined as the moment of maximum normalized amplitude. See Figure 6 shown.

Claims

1. A multiplication ratio fluorescent probe for epileptic focus localization, characterized in that: The integration consists of three components with complementary functions, and the general structure is as follows: in: The three components are: (1) PEG-b-PC7N, a pH-responsive core, responds sensitively to pathological pH changes; (2) PEG-b-PC7N-b-Cy5 and PEG-b-PC7N-b-Cy7, a FRET-based fluorescence pair, respond to ROS and H associated with neuroinflammation in lesions. + ; (3) RAP-PEG-b-PC7N is a targeting unit that promotes blood-brain barrier crossing and specific epilepsy targeting.

2. The method for preparing a multiplication ratio fluorescent probe according to claim 1, wherein: The specific steps are: (1) Preparation of PEG-b-PC7N; PEG-b-PC7N was prepared by adding PEG-CTA, 2-(azepan-1-yl)ethyl methacrylate, and AIBN at a molar ratio of PEG-CTA:2-(azepan-1-yl)ethyl methacrylate:AIBN=(5-8):(50-60):1 to a glass ampoule containing 1 mL of 1,4-dioxane and 1 mL of DMSO, degassing, sealing, and heating in an oil bath at 85-100°C to obtain PEG-b-PC7N. (2) Preparation of PEG-b-PC7N-b-Cy5 and PEG-b-PC7N-b-Cy7 fluorescent pairs; Synthesis of PEG-b-PC7N-b-Cy7: First, ACy7S was prepared by reacting K2CO3, allyl bromide, and compound 9: 3H-indole cation, 2-[2-[2-[(2-carboxyethyl)thio]-3-[2-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)ethylidene]-1-cyclohexen-1-yl]vinyl]-1,3,3-trimethyl in acetone at a molar ratio of K2CO3:allyl bromide:compound 9 = (1.1-1.3):(1.2-1.5):1 to obtain ACy7S; PEG-b-PC7N, ACy7 S, and AIBN were added to a glass ampoule containing 0.3-0.5 mL of 1,4-dioxane and 0.3-0.5 mL of DMSO at a molar ratio of PEG-b-PC7N:ACy7 S:AIBN = 2:(7-10):(0.0056-1) to react, and PEG-b-PC7N-b-Cy7 was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. Synthesis of PEG-b-PC7N-b-Cy5: Compound 3 (DR1-6) and triethylamine (TEA) were dissolved in 20-25 ml of anhydrous dichloromethane (DCM) at a molar ratio of (1-2):3.585, and stirred at -1-0°C for 30-35 minutes under nitrogen. Acryloyl chloride was then added dropwise at a molar ratio of compound 3:acryloyl chloride = 1:(2-2.5). The mixture was warmed to room temperature and stirred overnight. The solvent was then removed under reduced pressure, and the residue was purified by silica gel column chromatography to obtain ACy5 as a dark blue powder. PEG-b-PC7N, ACy5, and AIBN were added to a glass ampoule containing 0.3-0.5 mL of 1,4-dioxane at a molar ratio of PEG-b-PC7N:R-Cy5:AIBN=2:(7-10):(0.0056-0.007) and reacted using reversible addition-fragmentation chain transfer (RAFT) polymerization to prepare PEG-b-PC7N-b-Cy5. (3) Preparation of RAP-PEG-b-PC7N; CPADB-NHS, NH2-PEG 113 -Mal with CPADB-NHS:NH2-PEG 113 -Mal=(0.05~0.07)mmol:(0.06~0.08)mmol) was added to a round-bottom flask containing 3~5mL anhydrous CH2Cl2 to react and prepare CTA-PEG 113 -Mal; CTA-PEG-Mal, 2-(azepan-1-yl)ethyl methacrylate, and AIBN were added to a glass ampoule containing 1-3 mL of 1,4-dioxane at a ratio of CTA-PEG-Mal:2-(azepan-1-yl)ethyl methacrylate:AIBN = (0.004-0.006 mmol):(0.4-0.6 mmol):(0.0008-0.0010 mmol) to carry out RAFT polymerization to prepare PEG-b-PC7N-Mal. PEG-b-PC7N-Mal and RAP peptide were dissolved in PBS with a pH of 6.4 to 6.6 at a mass ratio of PEG-b-PC7N-Mal:RAP peptide = (20 to 25 mg): (4 to 6 mg). The two solutions were then mixed and N2 was bubbled through the mixture. The mixture was reacted at room temperature for 12 to 15 hours to prepare RAP-PEG-b-PC7N. (4) Finally, a pH-responsive type with targeted function was synthesized The method comprises the following steps: dissolving 1 to 2 mg of a pH-responsive polymer fluorescent donor PEG-b-PC7N-Cy5, 5 to 6 mg of a pH-responsive polymer fluorescent acceptor PEG-b-PC7N-Cy7, 0.05 to 0.10 mg of a RAP peptide-modified pH-responsive polymer RAP-PEG-b-PC7N, and 0.05 to 0.10 mg of a pH-responsive core PEG-b-PC7N in 1 to 2 mL of methanol; then, ultrasonically treating the mixture for 1 to 2 minutes; then, rapidly adding 9 to 12 mL of deionized water to the mixture, and continuing ultrasonic treatment for 10 to 15 minutes; evaporating the methanol at 40 to 50° C.; and washing the resulting nanoparticles with deionized water using a 100 kDa centrifugal filter tube and resuspending them three times, and then storing them at 4 to 5° C.

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