Synthesis of RBCQDs
RBCQDs were synthesized through a one-step hydrothermal method. In brief, 1 g of ginsenoside Rb1 was thoroughly mixed with 3 ml of anhydrous ethylenediamine in 10 ml of double-distilled water and stirred. The mixture was then transferred to a high-pressure reaction kettle with a polytetrafluoroethylene inner liner and subjected to hydrothermal synthesis at 200 °C for 24 h. The resulting solution was cooled to room temperature, and large particle impurities were removed by filtration through a 0.22 μm microporous membrane, yielding a brown transparent solution. In a light-avoiding environment, the solution underwent 12 h of dialysis purification using a membrane with a molecular weight cutoff of 1 kDa. The purified RBCQDs solution was then freeze-dried for storage.
Characterization of RBCQDs
High resolution transmission electron microscopy (Talos F200S G2, Thermo Scientific) was employed to analyze the morphology and size of RBCQDs with an acceleration voltage set at 200 kV. Fourier transform infrared spectrometry (Nicolet iS50, Thermo Scientific) was used to investigate the infrared absorption spectra of ginsenoside Rb1, anhydrous ethylenediamine, and RBCQDs, with samples prepared using a potassium bromide pellet method. X-ray photoelectron spectrometry (Nexsa G2, Thermo Scientific) was utilized to analyze ginsenoside Rb1 and RBCQDs with a 300 W Al K radiation source. The fluorescence spectrometer (F-4700, HITACHI) was employed to evaluate the 3D fluorescence characteristics of RBCQDs. NanoBrook 90Plus PALS (BROOKHAVEN) was used for determining the hydrodynamic diameter and potential of RBCQDs.
Preparation of Cy5-RBCQDs fluorescent probe
Following the methodology outlined in reference [34], the fluorescent probe Cy5-RBCQDs was synthesized by esterification reaction, coupling the hydroxyl groups of RBCQDs with the carboxyl groups on Cy5. In brief, RBCQDs and Cy5 were dissolved in N, N-dimethylformamide (DMF) solvent, thoroughly mixed, and 4-dimethylaminopyridine (DMAP) was added to the mixture. After reacting at room temperature for 24 h, 1,3-dicyclohexylcarbodiimide (DCC) was introduced, and the reaction continued in the dark for another 24 h. The reaction mixture was then poured into anhydrous ether, causing the product to precipitate, which was subsequently purified.
ABTS + clearance experiment
Following the manufacturer’s instructions, the liquid sample total antioxidant capacity Assay Kit (E2006, APPLYGEN) was utilized to assess the scavenging capacity of ginsenoside Rb1 and RBCQDs on free radicals. Solutions of ginsenoside Rb1 and RBCQDs with concentrations of 0, 0.1, 0.2, 1, 2, and 10 mg/mL were added to the ABTS + solution, thoroughly mixed, and then incubated at room temperature for 5 min. The absorbance of the solution was measured at 734 nm (A1). Absorbance values were obtained by replacing the sample with PBS (A0).
$${\rm{ABTS}}\,{\rm{Radical}}\,{\rm{Scavenging}}\,{\rm{Rate}}\left( \% \right) = \left[ {\left( {{\rm{A}}0 – {\rm{A}}1} \right)/{\rm{A}}0} \right)] \times 100\%$$
DPPH scavenging experiment
Following the manufacturer’s instructions, we used the DPPH free radical scavenging capacity assay kit (BC4755, SOLARBIO) to assess the scavenging capacity of ginsenoside Rb1 and RBCQDs on free radicals. Ginsenoside Rb1 and RBCQDs were briefly mixed with the DPPH solution and then incubated at room temperature for 30 min. The absorbance of the solution was measured at 517 nm (A1). Absorbance values were obtained by replacing the sample with PBS (A0). The DPPH radical scavenging rate (%)=[ (A0-A1)/(A0) ] × 100%.
Iron ion chelation experiment
To assess the iron chelation effect of ginsenoside Rb1 and RBCQDs, the fluorescence quenching degree and UV absorption spectral changes were compared before and after mixing with iron ions (Fe2+ or Fe3+). Specifically, Fe2+ or Fe3+ in the 0–1 mM range was added to the RBCQDs solution (3 mg/ml) and thoroughly mixed. The fluorescence emission spectra were measured using a fluorescence spectrometer at an excitation wavelength of 435 nm. For ginsenoside Rb1 and RBCQDs, solutions with 100, 200, 300, and 400 μg/ml were added to 1 mM Fe2+ or Fe3+ solutions. UV spectra of ginsenoside Rb1 and RBCQDs in the presence of iron ions were recorded using a multifunctional microplate reader, and the changes in the UV spectra were compared.
Cell culture
HT22 mouse hippocampal neuronal cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. All cells were maintained in an incubator at 37 °C with 5% CO2.
Cell viability assay
HT22 cells were randomly divided into four groups and seeded into a 96-well plate: Control group, RBCQDs group, Hemin group, and Hemin + RBCQDs group. Cells in the Control, RBCQDs, Hemin + CSF, and Hemin + RBCQDs groups were pretreated with 0, 25, 0, and 25 µg/ml of RBCQDs respectively for 12 h, followed by exposure to 0, 0, 100, and 100 µM of Hemin respectively for 24 h. Each well was then treated with 10 µl of CCK-8 solution. After a 4-hour incubation at 37 °C, the absorbance at 450 nm was measured.
Flow cytometry measurement of ROS
HT22 cells were seeded in a 6-well plate, randomized into different groups described above, and subjected to RBCQDs pretreatment and Hemin exposure. The intracellular ROS levels in other groups were detected using the DCFH-DA fluorescent probe. Flow cytometry measured and quantified the fluorescence intensity in the FITC channel of HT22 cells.
Non-heme iron ion detection experiment
Brain tissue samples were collected on the 3 days. In brief, according to the manufacturer’s instructions, the Iron Assay Kit (ab83366, Abcam) was used to detect the iron ion content in tissues.
Animal experiments
All mouse experiments were conducted following the ’Guidelines for the Care and Use of Laboratory Animals’ and approved by the Animal Ethics Committee of Nanchang University (Protocol Number: NCULAE-2,023,061,0001). Male C57BL/6J mice (8–10 weeks old) were used and obtained from Nanjing Kyurius Animal Co., Ltd. All mice were housed under standard conditions with a 12 h light/dark cycle (lights on at 6:00 AM, off at 6:00 PM), a temperature of 22 ± 1 °C, humidity > 30%, and ad libitum access to food and water.
ICH model and RBCQDs treatment
C57BL/6J mice (23-25 g) were induced with 2.5% isoflurane and maintained under anesthesia with 1.5% using a stereotaxic apparatus (68,537, RWD). Collagenase IV (67 U/ml, Sigma-Aldrich) was injected into the striatum at a dose of 0.04 µl/g (coordinates: 0.4 mm anterior to bregma, 2.2 mm right and 3.1 mm deep). A heating pad maintained the body temperature at 37 °C throughout the surgery. After the surgery, mice were provided ample food and water upon anesthesia recovery.
Mice were randomly divided into four groups: (1) Sham group: mice received intrathecal injection of 5 µl artificial cerebrospinal fluid (CSF,0.2 µl/g); (2) RBCQDs group: mice received intrathecal injection of RBCQDs (3 mg/ml) at a dose of 0.2 µl/g; (3) ICH + CSF group: mice underwent collagenase injection into the striatum and received an intrathecal injection of artificial CSF at a dose of 0.2 µl/g; (4) ICH + RBCQDs: mice underwent collagenase injection into the striatum and received an intrathecal injection of RBCQDs (3 mg/ml) at a dose of 0.2 µl/g.
Behavioral assessment
Before modeling and on days 1, 3, and 7 post-ICH, forelimb grip strength, left hindlimb pain threshold, and mNSS were assessed in mice (n = 6/group).
Frozen sectioning and HE staining
Tissues were fixed with paraformaldehyde for 48 h, dehydrated in 20% and 30% sucrose gradient, and sectioned into 15 μm slices using a cryostat (CM1952, Leica). For HE staining, tissues were stained with hematoxylin and eosin following the manufacturer’s instructions. Slices were imaged using an optical microscope (DMi8, Leica).
Observation of drug distribution
Observation of drug distribution was included in in vivo and ex vivo assessments. In vivo observation was conducted using an imaging system (IVIS, PerkinElmer) at different times: before intrathecal injection of Cy5-RBCQDs and 5 min, 30 min, and 3 h after injection. Ex vivo observation involved fixing and cryosectioning the brains of mice 30 min and 3 days after intrathecal injection of RBCQDs. The distribution of RBCQDs in the brain was observed using a slide scanner (VS120, Olympus) under an excitation wavelength of 488 nm.
Western blot analysis
Brain tissues from the ICH hematoma side were homogenized on ice. Purified proteins were quantified using the BCA protein assay kit. After SDS-PAGE gel electrophoresis and transfer to a PVDF membrane, the membrane was incubated with primary antibodies against MDA (ab27642), Cleaved caspase-3 (ab214430), and GAPDH (ab9485). Following secondary antibody incubation, chemiluminescence imaging was performed using the Tanon 5200 chemiluminescence system.
Immunofluorescence staining
As mentioned earlier, brain tissues were sectioned into 15 μm thickness frozen slices. The slices were incubated overnight with primary antibodies against MDA (ab27644) and Cleaved caspase-3 (ab214430). Subsequently, FITC and TRITC fluorescent secondary antibodies were used for 4 h. DAPI (ab285390) and Nissl (N21479) were used for neuronal staining.
Detection of brain tissue ROS levels
According to the manufacturer’s instructions, brain tissue ROS levels were measured using the Reactive Oxygen Species Assay Kit (C1300, APPLYGEN). In brief, brain tissues were prepared into single cell suspensions, and the DCFH-DA fluorescent probe was added to the cell suspension at a concentration of 10 µM. After incubating the cells at 37 °C for 30 min, centrifugation was performed at 1000 g for 5 min to collect cell precipitates. The precipitates were washed twice with PBS resuspended in PBS, and the fluorescence intensity was measured using a fluorescence spectrophotometer at an excitation wavelength of 488 nm.
Brain water content measurement
The brain water content was measured using the wet-dry weight method [35]. The calculation formula was [ (wet weight – dry weight)/(wet weight) ] × 100%.
Hematoma volume measurement
Following the protocol described in the literature [36], mouse brains were consecutively coronally sliced to quantify hematoma volume. The steps involved fixing the mouse brain in paraformaldehyde for 48 h and using a vibrating microtome (VT1200 S, LEICA) to cut the brain into 1 mm slices in the coronal plane. The hematoma volume was assessed by measuring the size of the hematoma in the brain using ImageJ software: hematoma volume = sum of hematoma areas × slice thickness.
Mouse forelimb traction test
The forelimb traction force recovery of mice was measured according to a published protocol [37]. Mice were placed steadily on a measuring board, and when the mice were calm, their tails were slowly pulled backward until they released their forelimbs. The instrument recorded the numerical value of the forelimb grip strength.
Mouse Hind paw pain threshold
The recovery of pain perception in mouse hind limbs was measured according to a published protocol [38]. Mice were placed in a measuring cage, and after the mice adapted to the surrounding environment, a fine needle was vertically placed on the sole of the mouse’s left hind paw. The needle was slowly lifted until the mouse lifted its left hind paw off the needle, and the maximum force applied by the needle during this process was read on the instrument as the mouse’s back paw pain threshold.
Modified neurological severity score (mNSS)
The recovery of mouse neurological function was comprehensively assessed using the mNSS [39]. The mNSS score comprises 10 tasks, including assessments of the mouse’s motor function (muscle status, abnormal movements), sensory function (visual, tactile, and proprioceptive sensation), balance, and reflexes. The scores range from 0 to 18, with higher scores indicating more significant neurological damage.
Cerebral meningeal blood Flow Measurement
Following the described procedure, the scalp was incised to expose the skull after anesthetizing the mice. The laser speckle blood flow imaging system (LSI, RWD) was used to measure the cerebral meningeal blood flow on the mouse brain surface. Each measurement was recorded for 10 s, and the average value was recorded.
Statistical analysis
ImageJ software was used to measure brain slice hematoma area and RBCQDs’ fluorescent distribution in brain tissues. Statistical analysis of experimental data was performed using GraphPad Prism 8 software. All data are presented as mean ± standard deviation. Independent sample t-tests or paired sample t-tests were conducted for comparisons between two groups, and one-way analysis of variance (ANOVA) with Turkey’s post hoc test was employed for comparisons among multiple groups. Two-way ANOVA with Turkey’s post hoc test was used to assess the effects of two factors. A significance level of P < 0.05 was considered statistically significant, and * represents p < 0.05.
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