Synthesis protocol of network nanocapsules with different proteins.
The synthesis protocol of network nanocapsules with different proteins was carried out by varying the pH to achieve optimal conditions. The aqueous solution of 5-HT and CAT/HSA samples were added to 21 mL Tris-HCl buffer (pH 9.5 or pH 7.4) to obtain inner-core nanoparticles (NPs). To obtain CAT-loaded nanocapsules (CNCs), the above inner NPs were incubated with HSA at a mass ratio of 1:1 in PBS for 1 h. Unabsorbed HSA was removed by membrane dialysis (100 kDa) in ultrapure water at pH 7.4 for 6 h. The size distribution of hydrodynamic diameters N(dh) of NPs from different pH buffers was characterized by dynamic light scattering (DLS) using a UV-cuvette (ZH 8.5mm; Deckel; Sarstedt, Germany). The distribution of ζ-potential I(ζ) of these NPs was also recorded by DLS with laser Doppler anemometry (LDA). The optimal pH value was found to be 9.5. After performing the optimized synthesis protocol, the load capacity of different proteins was tested. Four different proteins were chosen, namely bovine serum albumin (BSA, Aladdin, #A104912, China), catalase (CAT, Aladdin, #C128526, China), α-chymotrypsin (α-CT, Sigma-Aldrich, #C4129, Germany), and lysozyme (Lys, Aladdin, #L105521, China), each with different molecular weights and isoelectric points. Nanocapsules loaded with proteins were constructed using the above protocol. Since the synthetic procedures contained two proteins (HSA and cargo proteins), the cargo proteins were conjugated with fluorescein5(6)-isothiocyanate (FITC, Aladdin, #F106837, China) to quantify the loading efficiencies. The samples were purified and concentrated by lyophilization. The labeling efficiency was calculated using Beer-Lambert’s law through ultraviolet visible spectrum (UV-vis, UV-2450, Shimadzu, Japan). Furthermore, the nanocapsules loaded with fluorescent proteins were synthesized using the same methods. The fluorescence intensity IFITC at 520 nm emission wavelength under excitation at 488 nm of proteins-FITC was recorded by a fluorescence meter (HORiBA Fluoromax-4, Horiba Scientific, Japan) to quantify the loading efficiencies.To achieve brain targeting ability, rabies virus glycoprotein (RVG29) was used for the surface functionalization of NPs. CNCs were incubated with RVG29 at a mass ratio of 4 eq of CNCs per 1 eq of RVG29 in PBS for 1 h to obtain virus-inspired CAT-loaded nanocapsules (VCNCs). Unabsorbed RVG29 was removed by membrane dialysis (5 kDa) in ultrapure water at pH 7.4 for 4 h. For the control group (virus-inspired nanocapsules (VNCs)), only HSA (1500 µL) was used for synthesis. The hydrodynamic diameters dh(N) and ζ-potential values of the samples during the synthetic procedures were measured by DLS and LDA, respectively. The shape of VCNCs and CNCs was determined using transmission electron microscopy (TEM, Talo F200S SUPERX, Thermo Scientific, USA). To obtain TEM images, a droplet of the sample was placed on top of a copper grid. The synthesis protocol for the inner core NPs and the FITC labeling protocol are detailed in the supporting information section.
Structural characterizations of 5-HT covalent bonds.
To confirm the formation of 5-HT covalent bonds, the structure was characterized using mass spectrometry, UV-vis spectra, and Fourier Transform Infra-Red (FTIR, Nicolet is 50, Thermo Scientific, USA). The 5-HT covalent bonds were synthesized using the aforementioned methods without the addition of proteins. The structure was measured using Electrospray Ionization Mass Spectroscopy (ESI-MS, LTQ Orbitrap Velos Pro, Thermo Scientific, USA). Additionally, CAT, HSA, 5-HT, VCNCs, CNCs, and VNCs at a concentration of C = 1 mg/mL were characterized using UV-vis spectroscopy and FTIR spectroscopy.
Characterizations of VCNCs, CNCs, and VNCs.
The morphology of the nanocapsules was characterized using DLS and TEM. The colloidal stability of the nanocapsules was studied using DLS in PBS. Specifically, 1 mg of nanocapsules was mixed with 1 mL of PBS, and the hydrodynamic diameter dh(N) and ζ-potential were measured using DLS over a total period of 12 d. The enzyme stability of CAT in CNCs and VCNCs was evaluated in PBS over a period of 5 d. For this, VCNCs, CNCs, and free CAT were dissolved in 1 mL of PBS at a concentration of CCAT = 10 µg/mL each and were kept at room temperature for 5 d. The CAT viability was measured at t = 0 h as a control using a testing kit (Solarbio, #BC0200, China). After the specific incubation times, the data were measured and normalized to V = 100% at t = 0 h. The data are presented as viability percentages. To explore the anti-adsorption properties of unspecific proteins, the interaction between the nanocapsules and bovine serum albumin (BSA) was investigated. Briefly, 0.5 mL of VCNCs, CNCs, and VNCs at a concentration of CNPs = 1 mg/mL was incubated with 0.5 mL of BSA at a concentration of CBSA = 1 mg/mL on a shaker for 24 h. At specific time points, the free proteins were separated using an ultracentrifuge filter (cutoff molecular weight 100 kDa) at 2000 rpm for 10 min. The eluents were collected and tested using BCA assays according to the instructions (Beyotime, #P0010, China). The data were normalized using the sham group (BSA alone). To quantify the 5-HT concentration in the nanocapsules, the UV-vis spectra of different dilutions of 5-HT were measured, and the UV-vis absorbance at 204 nm versus the 5-HT concentration was fitted with a linear function. The absorbances of VCNCs, CNCs, and VNCs at 204 nm were extracted to calculate the concentration of 5-HT. Next, the protein concentrations in the nanocapsules were measured using a BCA assay (Beyotime, #P0010, China), and the CAT concentration was measured using a testing kit (Solarbio, #BC0200, China) according to the instructions.
Stimuli-responsive release of 5-HT.
To simulate the real environment, we tested the release of 5-HT in PBS buffer (pH 7.4), citric acid-Na2HPO4 buffer (pH 6.4), pH 7.4 + ROSUP buffer, and pH 6.4 + ROSUP buffer (1:1000, Beyotime, #S0033S, China). Initially, we characterized the morphology changes of nanocapsules in acidic and inflammatory conditions. We immersed 1 mg of VCNCs, CNCs, and VNCs respectively in 1 mL of pH 6.4 buffer (Citric acid-Na2HPO4 buffer), pH 7.4 + ROSUP buffer, and pH 6.4 + ROSUP buffer for 8 d. At specific time points, we tested the mean hydrodynamic diameter dh(N) by dynamic light scattering (DLS). We also measured the nanocapsule degradation using high-performance liquid chromatography (HPLC, Agilent 1260 Infinity, Agilent Technologies, USA) and ESI. VCNCs, CNCs, and VNCs at a concentration of C5 − HT = 5000 µg/mL were dissolved in different media, namely pH 7.4 (PBS), pH 6.4 buffer, pH 7.4 + ROSUP buffer (1:1000), and pH 6.4 + ROSUP buffer (1:1000) for 72 h. The mixture was then placed in an ultracentrifuge tube with a 3 kDa cutoff and centrifuged at 8000 rpm for 30 min. The remaining NPs and free proteins were retained in the filters, while the liberated 5-HT monomers were eluted. We collected the eluate and analyzed it by HPLC combined with ESI-MS. Next, we collected the separated samples at retention time from 2 to 4 min and tested them by ESI-MS. To quantify the cumulative release profile of 5-HT, we measured the eluate by HPLC. To demonstrate that CAT protects the structure of 5-HT under oxidative conditions, we incubated 150 µg/mL of 5-HT with CAT (2 µg/mL) for 1h in pH 7.4 + ROSUP buffer. The control groups represented the same concentration of 5-HT and CAT, respectively, in pH 7.4 + ROSUP buffer for 1 h. Additionally, 150 µg/mL of 5-HT was incubated in ultrapure water for 1 h. After incubation, samples were centrifuged with ultracentrifuge tubes with a cutoff molecular weight of 3 kDa at 8000 rpm for 30 min. The eluent was characterized by UV-Vis spectra. The methods of HPLC are shown in the supporting information.
Fluorescence labeling.
Human serum albumin (HSA, Aladdin, #H304436, China) was labeled with Cy5.5 NHS (Aladdin, #C171354, China) and Cy7.5 NHS (Aladdin, #C171364, China), and rabies virus glycoprotein (RVG29, Glbiochem, China) was labeled with FITC (Aladdin, #F106387, China). The labeling process is described in detail in the supporting information. HSA-Cy5.5 or HSA-Cy7.5 and RVG29-FITC were used to construct fluorescence-labeled nanocapsules using the method described above.
Cell Cultures And Cytotoxicity Assays
Mouse brain cell line bEnd.3 cells and rat pheochromocytoma cell line PC-12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Excell, China) and 100U/mL penicillin/streptomycin (Gibco, USA) at 37°C and 5% CO2.
The resazurin assay was used to evaluate the cell viability of bEnd.3 and PC-12 cells after exposure to VCNCs, CNCs, VNCs, CAT, 5-HT, and HSA. PC-12 and bEnd.3 cells were seeded in 96-well plates with 0.32 cm2 growth area per well at a density of 30,000 cells in 150 µL medium per well. After overnight growth, the cells were exposed to different samples for 24 and 48 h. The concentration ranges of all the samples are shown in Table S4. After incubation, the cells were washed once with 150 µL of PBS. Then, 100 µL of 10% resazurin solution (0.25 mg/mL) in complete cell media was added to the cells and incubated for 4 h. The OD value of each well was measured at 570 nm and 600 nm using a microplate reader (Epoch, BioTek, USA). The OD value is positively related to the number of living cells. The OD values of cells incubated with test samples were normalized to the cell medium containing resazurin which had not been exposed to cells and samples. The cell viability was calculated using Eq. 1 and Table S4. Eq. 1: %Reduction of resazurin agent = (Eoxi600 × A570) - (Eoxi570 × A600) / (Ered570 × C600) - (Ered600 × C570) Where Eoxi570 = molar extinction coefficient of oxidized resazurin agent at 570 nm = 80586; Eoxi600 = molar extinction coefficient of oxidized resazurin agent at 600 nm = 117216; Ered570 = molar extinction coefficient of reduced resazurin agent at 570 nm = 155677; Ered600 = molar extinction coefficient of reduced resazurin agent at 600 nm = 14652; A570 = absorbance of samples at 570 nm; A600 = absorbance of samples at 600 nm; C570 = absorbance of negative controls (cell medium with resazurin agent) at 570 nm; C600 = absorbance of negative controls (cell medium with resazurin agent) at 600 nm.
Endocytosis studies using flow cytometry.
In this study, we employed flow cytometry (BD LSR Forteassa Biosciences, USA) to analyze the cellular uptake of VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs-Cy5.5. Initially, bEnd.3 cells were seeded at a density of 150,000 cells/mL in a volume of 1 mL into 24-well plates (growth area = 1.9 cm2) and were allowed to incubate overnight. Subsequently, the cells were incubated with fresh cell medium containing Cy5.5 labeled nanocapsules at a concentration of C5-HT = 25 µg/mL for 1, 3, and 5 h. Time-dependent endocytosis of nanocapsules was monitored by characterizing the cellular fluorescence over time using flow cytometry. At specific time points, the supernatant was removed, and cells were washed twice with 1 mL of PBS. The cells were then detached by adding 0.1 mL of 0.05% trypsin/EDTA solution (Procell, China) and neutralized with 0.4 mL of complete cell medium. We collected the cells by centrifugation at 400 g for 8 min, and the obtained cell pellet was resuspended in 0.2 mL of PBS and analyzed using flow cytometry (BD LSR Forteassa Biosciences, USA). For each experiment, 20,000 gated cells were counted and analyzed.
In vitro model for detecting the penetration efficiency of nanocapsules.
PC-12 cells and bEnd.3 cells were used to construct an in vitro blood-brain barrier (BBB) penetration model. Briefly, bEnd.3 cells were seeded onto transwell filters (pore size 0.4 µm, 6.5 mm, Costar, USA) at a density of 80,000 cells/well. Meanwhile, PC-12 cells were seeded into 24-well plates (surface area 1.9 cm2) with 120,000 cells per well in 1 mL of complete cell medium. It's important to note that bEnd.3 cells and PC-12 cells were not in the same well. The next day, the cells were exposed to 0.3 mL of VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs-Cy5.5 (C5 − HT = 25 µg/mL) in complete cell medium for an exposure time of texp = 3 h. After that, one part was used for the analysis of the uptake study, characterized by flow cytometry. The other part was used for further study. The supernatant was replaced by washing with PBS twice, and the cells were further incubated with fresh medium. The insert filters containing bEnd.3 cells were further coincubated with PC-12 cells at the lower chamber for another incubation time of tinc = 24 h to characterize the penetration of nanocapsules from the apical chamber to the basolateral chamber. The intracellular fluorescence of collected cells in the apical chamber and basolateral chamber was analyzed by flow cytometry to calculate the penetration efficiency at texp + tinc = 3 h + 24 h. The intracellular fluorescence per cell decreases over time due to proliferation. Cell proliferation needs to be taken into account when studying permeability efficiency. Therefore, the number of cells in every testing sample was counted. The normalized penetration efficiencies were calculated from the data in Fig. S24 and Fig. 3A, multiplied by the growth factor in Fig. S25. The penetration efficiency was also visualized by confocal laser scanning microscopy (CLSM; LSM900, Zeiss, Germany). At texp + tinc = 3 h + 24 h, the PC-12 cells and bEnd.3 cells were fixed with 4% paraformaldehyde (100 µL/well), washed twice with PBS, and stained with DAPI (Beyotime, #C1006, China) at 37°C. The images of the samples were finally captured under CLSM. To further study the integrity of nanocapsules after passing through bEnd.3 cells, dual-labeled nanocapsules (HSA-Cy5.5, RVG29-FITC) were adopted to establish the transwell system. The methods were the same as the ones using Cy5.5 labeled nanocapsules.
Cellular Anti-inflammation Of Nanocapsules After Passing Through Bend.3 Cells
To assess the anti-inflammatory properties of nanocapsules after passing through bEnd.3 cells, we investigated their ability to scavenge ROS. 2’,7’ –dichlorofluorescein diacetate (DCFH-DA) is commonly used to detect intracellular ROS levels. To induce intracellular ROS production, we added the ROSUP reagent (1:1000, Beyotime, #S0033S, China) to PC-12 cells in serum-supplemented medium for 0.5 h. The cells were pre-incubated with bEnd.3 cells containing nanocapsules in the transwell filter for texp + tinc = 3 h + 24 h. The PC-12 cells were then washed twice with PBS and incubated with 0.5 mL of 0.1% DCFH-DA (Beyotime, #S0033S, China) diluted in serum-free DMEM medium for 0.5 h at 37°C. The cells were then washed twice with PBS and exposed to 0.5 mL of FBS-free DMEM medium. The fluorescence of DCFH was measured using a microplate reader (Envision@2015, PerkinElmer, USA) and an inverted fluorescence microscope (Eclipse Ti2, Nikon, Japan) at excitation and emission wavelengths of 488 nm and 520 nm, respectively. To investigate whether the covalent bond between 5-HT and phenol influences ROS concentration, we performed additional experiments using the same methods as described above. Further details of these experiments are provided in the supporting information.
Cytoplasmic calcium concentration assay.
To assess changes in cytoplasmic calcium concentration, we used Fluo-4 AM (Beyotime, #S1060, China). We seeded 150,000 PC-12 cells in 1 mL of cell culture medium with 10% FBS in a 24-well plate (growth area 1.9 cm2) and incubated them overnight. The cells were then exposed to different concentrations of VCNCs, CNCs, VNCs, 5-HT, CAT, and HSA for 3 h, as specified in Table S5. After exposure, we washed the PC-12 cells twice with 1 mL of PBS and stained them in 0.5 mL of FBS-free DMEM medium containing 0.2 µM Fluo-4 AM for 0.5 h. The cells were then washed twice with PBS and kept stained for another 0.5 h. The fluorescence of Fluo-4 was detected using a microplate reader (Envision@2015, PerkinElmer, USA) at excitation and emission wavelengths of 488 nm and 520 nm, respectively.
Cellular release of 5-HT monomers from nanocapsules.
To investigate the cellular release of 5-HT monomers from nanocapsules, PC-12 cells were seeded in 24-well plates with a surface area of 1.9 cm2 at a density of 150,000 cells per well in a volume of 1 mL. The following day, pH 6.4 medium was prepared by mixing 1M HCl and serum-supplemented medium to obtain the medium at pH 6.4. The ROSUP medium was prepared by diluting 1 µL of ROSUP reagent (Beyotime, #S0033S, China) with 1 mL of complete DMEM medium. Cells were incubated with VCNCs, CNCs, and VNCs at a dose of C5 − HT = 25 µg/mL in complete DMEM media, pH 6.4 media, ROSUP media (1:1000), and pH 6.4 + ROSUP media for 3 h. After incubation, the supernatant above the cells was collected, and the cells were washed twice with PBS. The cells were detached from the 24-well plate using 0.05% trypsin/EDTA in a volume of 0.1 mL. After aspirating the trypsin, 0.3 mL of medium was added to collect the cell pellets, which were then centrifuged at 400 g for 8 min and washed twice with PBS to obtain the cell pellet. Each sample was added to 0.1 mL of PBS, and a handheld homogenizer was used to crumb the cell samples. The supernatant and cell samples were then analyzed using a 5-HT Elisa Kit (Elabscience, #E-EL-0033c, China), following the manufacturer's instructions.
Behavioral tests and chronic unpredictable mild stress (CUMS) model.
Male C57bl/6j mice (3–4 w) were obtained from Hunan SJA laboratory animal Co., Ltd (Changsha, China) and were housed at a constant temperature of 21°C and 50% humidity. All procedures were approved by the Committee on Experimental Animal at XiangYa Hospital, Central South University (grant number: 2022020460). After one week of habituation, behavioral tests were used to select mice with normal locomotion. Subsequently, the mice were subjected to different and repeated unpredictable stressors for five weeks to establish the CUMS model. The CUMS-induced depressed mice were exposed to various mild stressors that changed from day to day to establish an unpredictable procedure. The stressors involved changes in the environment (reversed light/dark cycle, restraint, cage tilting, strobe light, wet bedding), social stressors (crowding), fear stressors (cold swim, tail pinch), and water/food deprivation. A detailed account of the procedures in the five weeks is provided in Table S6. Behavioral tests were used to confirm the establishment of the CUMS depression model. Subsequently, the mice were randomly divided into six groups (n = 6): 0.9% saline (CUMS + saline), 400 µg/kg fluoxetine (CUMS + Flu), C5 − HT: 400 µg/kg VCNCs (CUMS + VCNCs), C5 − HT: 400 µg/kg CNCs (CUMS + CNCs), C5 − HT: 400 µg/kg VNCs (CUMS + VNCs) via intravenous injection at 2-day intervals for two weeks. To maintain the depressed state, the mice continued to be subjected to stressors during the treatment session (Table S7). Finally, the therapeutic outcomes were measured using three behavioral tests: the sucrose preference test (SPT), open field test (OFT), and forced swim test (FST). The results were analyzed using Smart V03 software. The details of the behavioral tests are provided in the supporting information.
Blood collections and measurements.
After the third-session behavioral tests, blood was drawn from the mice's eyeball. One part of the blood was collected in heparin anticoagulant tubes, and the blood panel parameters were measured using an Automatic Analyzer. Another part of the blood was left undisturbed at room temperature for 30 min to allow it to clot. The samples were then centrifuged at 4000 g for 10 min at 4°C to remove the clot and collect the serum. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in the serum were analyzed using an Automatic Analyzer. The serum cortisol level was characterized using the ELISA kit (Elabscience, #E-EL-0161c, China) according to the manufacturer's instructions.
In vivo brain targeting ability.
To investigate the brain targeting ability of the nanocapsules, we intravenously injected Cy7.5-labeled nanocapsules at a dose of C5 − HT: 400 µg/kg into male C57bl/6j mice (20-25g) and CUMS mice for 3 h. To minimize interference from fur, a large portion of the mice's fur was removed before injection. We used the IVIS Lumina II (Perkin Elmer, USA) to observe the in vivo fluorescence of Cy7.5. Subsequently, we harvested the brains for ex vivo fluorescence imaging. To further validate our findings, we conducted a similar study on nude mice (4 w) that received intravenous administration of Cy5.5-labeled nanocapsules at C5 − HT: 400 µg/kg. After 3 h of injection, we used the IVIS Lumina II to observe the in vivo fluorescence.
Histological Assays
After blood collection, one part of the mice was perfused with PBS and 4% paraformaldehyde, and the brains were harvested, embedded in paraffin and OCT compound, and sectioned into 4 µm and 9 µm thickness, respectively. Other parts of the mice were sacrificed after blood collection, and major organs (heart, liver, spleen, lung, and kidney) and brain were harvested. The hippocampal regions were dissected from the brain on ice and stored at -80°C. The slices were processed with Hematoxylin-eosin staining (HE), immunohistochemistry (IHC), Nissl, and ROS fluorescent staining. Details are provided in the supporting information.
The measurements of biochemical factors in the hippocampus.
For the enzyme-linked immunosorbent assay (ELISA) study, the dissected hippocampus was added to PBS and homogenized for 1 min. The sample solutions were then measured using BDNF, TNF-α, and 5-HT ELISA kits (Elabscience, #E-EL-M0203c, #E-EL-M3063, and #E-EL-0033c China) according to the manufacturer’s instructions. For reverse transcription quantitative polymerase chain reaction (RT-qPCR), the total RNA of the hippocampus was extracted with TrizolTM Reagent (Life Technologies, Carlsbad, USA), and PCR amplification was performed using GoScriptTM Reverse Transcription System (Promega, #A5000, USA). The sequences of primers are provided as follows. The RT-qPCR was analyzed using GoTaq@ qPCR Master Mix (A6001, Promega, USA). The relative mRNA expression of target genes was monitored by QuantStudio 6 Flex (Applied Biosystems, Life Technologies, USA) with the 2−△△Ct method. Endogenous GAPDH was adopted as the negative control. The primer pairs were as follows: GAPDH, forward 5’-GCCAAGGTCATCCATGACAACT-3’, reverse 5’-GAGGGGCCATCCACAGTCTT − 3’;BDNF, forward 5′-CAGGGGCATAGACAAAAG-3’, reverse 5’-GAGGGGCCATCCACAGTCTT − 3’; NLRP3, forward 5’ -CTCGCATTGGTTCTGAGCTC-3’,reverse 5’-AGTAAGGCCGGAATTCACCA-3’..
mRNA sequencing analysis.
Total RNA was extracted from the hippocampus of mice brains using TrizolTM Reagent (Life Technologies, Carlsbad, USA), and mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase. Subsequently, second-strand cDNA synthesis was performed using DNA Polymerase I and dNTP. The AMPure XP system (Beckman Coulter, Beverly, USA) was used to select cDNA fragments with a length of 370–420 bp for PCR amplification. After PCR procedures, the product was purified by AMPure XP beads to obtain the library. The quality of the library was tested using the Qubit2.0 Fluorometer and Agilent 2100 bioanalyzer. After confirming that the insert size met the expectation, the concentration of the library was quantified using qRT-PCR. The different libraries were pooled according to the effective concentration and the target amount of data off the machine, and then sequenced using the Illumina NovaSeq 6000. For differential expression analysis, the read counts were normalized using the DESeq2 program through the scaling factor. P values were adjusted using the Benjamini & Hochberg method. A padj ≤ 0.05 and |log2(fold change)|≥1.5 were set as the threshold for significantly differential expression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Gene Ontology (GO) were used to identify enriched pathways through the clusterProfiler R package (3.8.1). Gene Set Enrichment Analysis (GSEA) is a computational approach to determine significant differences between two samples. The genes were ranked to check for enriched genes at the top or bottom of the Gene Set list.