Reagents

Curcumin, PLGA (75: 25) purchased, polyvinyl alcohol (PVA) purchased from Macklin Company (Shanghai, China). 2’,7’-Dichlorodihydrofluorescein diacetate, (DCFH-DA) purchased from LABLEAD (Beijing, China). MPTP and 1-Methyl-4-phenylpyridinium iodide (MPP+) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The apoptosis kit, mitochondrial membrane potential detection kit, autophagy kit, cell counting kit (CCK8), 4’,6-diamidino-2-phenylindole (DAPI) obtained from Beyotime Biotechnology (Shanghai, China). All antibodies including TH, IBA-1, TNF-α, VDAC1 and conjugated secondary antibodies were bought from Servicebio (Wuhan, China). Sulfo-Cyanine7 NHS (Cy7) were acquired from MeilunBio (Dalian, China).

Microfluidic chip design and fabrication

All microfluidic chip channels were set to 1 mm in height and 1 mm in width. The circular incubator was 1 mm high and 4 mm in diameter. The aluminum electrode was placed in the electroporation area under the PC channel with a width of 10 mm and a thickness of 0.1 mm to ensure the electroporation effect of the fluid in the channel. Using standard soft lithography technology, a silicon wafer mold with designed pattern was made using photoresist. The aluminum electrode was fixed to the glass substrate by soft lithography and thermal evaporation. The PC layer was then cast from the silicon mold, punched holes, and finally bonded to an electrode-patterned glass substrate using an oxygen plasma system (Harrick Scientific, USA). All hot bonded form a closed flow channel. Finally, the device was heated at 120℃ for 2 days to ensure soundness and regain hydrophobicity.

Preparation and characterization of MM-Cur-NPs

PLGA nanoparticles were prepared by water/oil/water-liquid solvent evaporation technology. PLGA was dissolved in acetone and then emulsified by phacoemulsification in 1% (w/v) PVA in water to produce a primary emulsion. The above emulsion then added to an aqueous solution containing 1% (w/v) PVA, and then sonicated again to form a secondary emulsion. Centrifuge and wash twice with deionized water. After drying, the nanoparticles were redispersed in acetone and slowly dripped into the curcumin solution. The nanoparticles were redispersed in 2 mL of deionized water by centrifugation at 15, 000 rpm, 20 min, 4℃ and washed twice with deionized water. Neuro-like cells were harvested with reference to our previous study [7]. Suspend cells in homogenization buffer containing Tris-HCl (pH = 7.5), KCl, sucrose, MgCl2, and protease/phosphatase inhibitors. The obtained mixture was crushed with a homogenizer IKA (T18) homogenizer. Centrifuge sequentially at 3, 000 g for 10 min and 10, 000 g sequentially at 4℃ for 30 min to obtain the suspension. The polyethylene tube was slowly introduced into the microfluidic chip by syringe pump (TS2 − 80, Longer Precision Pump, China) with 500 mg of PLGA nanoparticles and 1 mL of cell membrane for complete particle coating, respectively. The electrical pulse parameters refer to past reports and optimize the voltage and duration of the electrical pulse and the flow rate of the solution to achieve effective electroporation. When the duration and flow rate were 200 µs and 20 µL·min− 1, respectively, the voltage was regulated between 20 and 70 V. When the voltage and flow rate were 50 V and 20 µL·min− 1, respectively, the duration was adjusted from 50 µs to 300 µs. When the voltage and duration were 50 V and 200 µs, respectively, the flow rate was regulated in the range of 10–50 µL·min− 1. After electroporation, the resulting mixture was collected from the outlet and sonicated.

Characterization of MM-Cur-NPs

The nanoparticle solution was droppedly onto the copper mesh, and after 30 min of sample deposition, rinse the grid twice with PBS. Add 5 µL of uranyl acetate stain to the nanoparticle-loaded grid. The morphology of these nanoparticles was characterized using transmission electron microscopy (TEM, JEOL, JEM-6700 F) applying an accelerating voltage of 120 KV. Dynamic light scattering (DLS) was used to determine the particle size of nanoparticles.

Cell culture and viability

Human neuroblastoma cells SH-SY5Y, microglial cells BV2 were seeded in DMEM medium supplemented with 10% fetal bovine serum (FBS, BI, Israel), 1% 10,000 units/mL penicillin and 1% 10 mg/mL streptomycin (BI, Israel), and 100 µmol L-ascorbic acid (BI, Israel). Place the flask (Corning, USA) in a constant temperature incubator at 37℃ with 100% humidity and 5% CO2 concentration. Replace with fresh medium every 2–3 days and passage after a cell confluency of 80–90%. The in vitro models of PD constructed by pretreating SH-SY5Y cells and BV2 cells with MPP+ (4 mM) for 24 h, the effect of nanoparticles on cell viability was detected by CCK8 colorimetry. A total of 2 × 105 cells/mL of SH-SY5Y cells and BV2 cells were seeded into 96-well plates, incubated at 37℃ for 1 h with CCK8 solution after 24 h of different drug treatment, and finally measured by microplate reader the absorbance generated at 450 nm. A total of 3 independent experimental replicates were performed.

Detection of autophagy

SH-SY5Y and BV2 cells were seeded in 6-well plates, respectively, and after MPP + treatment for 24 h, different drugs were added for 24 h. Remove the medium, add MDC staining solution, incubate at 37 °C protected from light for 30 min in a cell culture incubator, wash 3 times with Assay Buffer, and measure the fluorescence intensity of each sample with fluorescence microscopy (EVOS, Thermofisher, USA).

Reactive oxygen species analysis

SH-SY5Y and BV2 cells were seeded in 6-well plates, respectively, and after MPP + treatment for 24 h, different drugs were added for 24 h. Remove the medium, add 20 µM DCFH-DA staining solution and incubate at 37℃ for 30 min, wash 3 times with basal medium, and measure the fluorescence intensity of each group with fluorescence microscopy (EVOS, Thermofisher, USA).

Mitochondrial membrane potential analysis

SH-SY5Y and BV2 cells were seeded in 6-well plates, respectively, and after MPP + treatment for 24 h, different drugs were added for 24 h. Remove the medium, add JC-1 staining solution and incubate at 37℃ for 30 min, wash 3 times with basal medium, and measure the mitochondrial membrane potential (MMP) fluorescence intensity of each group with fluorescence microscopy (EVOS, Thermofisher, USA).

Detection of membrane proteins

Free vesicles or proteins were first removed from the sample solution by centrifugation and unbound proteins were removed by dialysis with a 30 nm porous membrane for 12 h. Proteins from mesenchymal stem cell-derived neuron-like cell membranes, cell membrane-coated nanoparticles, and individual nanoparticle groups were extracted through 95℃ for 5 min. After cooling at room temperature, 25 µL of sample from all groups was loaded into each well of the Bio-Rad electrophoresis system. Protein staining was done bright blue using Coomassie and destained with acetic acid overnight prior to imaging.

Animals and treatment

The male C57BL/6 mice (6–8 weeks) were purchased from Charles River (Beijing, China). Animal experiments was carried out in accordance with the ARRIVE guidelines, and the China Academy of Chinese Medical Sciences Animal Care and Use Committee ethical standards and national guidelines. All procedures were approved by the Ethics Committee of China Academy of Chinese Medical Sciences for the use of experimental animals. All mice were maintained under static environment with a temperature of 22℃ ± 2℃, a humidity of 55-65%, and a light/dark cycle of 12 h, water and food were provided free of charge, and all animals were euthanized after the completion of the experiment. All mice were randomly divided into 5 groups (male, 6-8 weeks, n = 6), including control group, PD group, curcumin group, curcumin-PLGA nanoparticles group and MM-Cur-NPs group. MPTP-induced mouse models of PD were fabricated by daily intraperitoneal injection of MPTP (25 mg/kg/d) for 7 consecutive days. Group of Curcumin (Cur, 100 mg/kg/2d), curcumin-PLGA nanoparticles (Cur-NPs, 100 mg/kg/2d), MM-Cur-NPs (100 mg/kg/2d) were administered through the nasal cavity at a dose of for 4 weeks. Behavioral testing of all grouped mice was performed one week after the end of treatment.

Behavioral analysis

All mice were performed to behavioral testing to assess the mobility function of mice. The following three behavioral experiments were tested:

Rotation rod test: Using the accelerated rotation device, train mice in accelerated mode (4-40 rpm) for three min continuously for three days before testing. Train at a constant rotational speed (16 rpm) until mice can stay on the rod for at least 100 s. Perform a formal experiment by placing mice on a rotating roller and speeding up from 4 rpm to 40 rpm in 3 min. The time of the first drop of the mouse is recorded to represent the length of time the mice stay on the rod. All mice were tested 3 times.

Pole test: A rough wooden pole with a diameter of 16 mm and a height of 60 cm was made, placed on the top of the mouse, and the time it took to climb to the bottom was recorded. All mice were kept for training starting 3 days prior to testing, and all mice were tested 3 times.

Suspension test: Place a thin horizontal line 40 cm from the table, pick up the mouse, and hang both front paws on the line. Two paws can be grasped as 3 points, single paws can be grasped as 2 points, and if they cannot be grasped, they were counted as 1 point. All mice were kept for training starting 3 days prior to testing, and all mice were tested 3 times at the time of formal trials.

Distribution detection of nanoparticles

The fluorescent probe, Cy7, was used to evaluate the brain-targeted efficiency of MPTP to treat nanoparticles in PD mice. Three group Cy7, Cur-NPs, and MM-Cur-NPs were given nasally to assess the biodistribution of brain. Animals were imaged at 0, 1, 3, 6, 12, 24 h after administration in vivo, using Living Image software (Caliper, Alameda, CA).

Detection of inflammatory factors

All mouse blood was allowed to stand and centrifuge (4℃, 3000 rpm, 20 min) to obtain serum. We refer to the manufacturer’s protocol of ELISA kits from Jiangsu Meimian Industrial Co., Ltd (Jiangsu, China). All serum levels of IL-4, IL-10, IL-6, TNF-α were measured.

Histological analysis

Histochemical analysis was performed 5 weeks after the start of the first nanoparticle treatment. For histopathological analysis, tissue samples (lung, liver, kidney, spleen, heart, and brain) were collected immediately after euthanasia in all mice. Samples were routinely fixed in 10% buffered formalin, embedded in paraffin, and sectioned approximately 5 μm. Lung, liver, kidney, spleen, heart were used to stain with common hematoxylin and eosin (H&E). Microscopic study of tissue sections by light microscopy (Olympus-CH30, Japan) to identify possible histopathological lesions. All mouse brain tissue was sliced into a coronal plane. Striatum and substantia nigra regions were determined by serial slices with reference to brain region coordinates. Midbrain tissue was used to detect TH in the substantia nigra by conjugating secondary antibody with HRP and developing color by DAB. The striatum of the midbrain was individually embedded for the detection of TH, IBA-1, TNF-α, and VDAC1 by conjugated secondary antibodies with fluorescent dyes. The high-resolution immunofluores cence results were scanned and saved using a confocal microscope (Leica Biosystems). The intensity of the fluorescence was detected in the chemiluminescence analyzer and analyzed with ImageJ software.

Microdialysis analysis

On the third day after modeling, the PD mice were anesthetized by inhalation of 2.0% isoflurane (flow rate 0.5 L·min− 1), and under the guidance of stereotaxic instrument, probe cannula was embedded in mice striatum (2.2 mm before the front halogen, 1.5 mm next to the middle seam, 2.25 mm into the needle) and fixed with dental cement. Place mice in a freely mobile device (33 cm× 40 cm× 36 cm) and perfuse 2 probe pathways with modified Ringer solution overnight at a flow rate of 0.3 µL·min− 1. The next day, adjust the flow rate to 1.3 µL·min− 1, equilibrate for 1.5 h, and start collecting the dialysate. MM-Cur-NPs were then administered nasally and the dialysate was collected continuously at the same volume and frequency. Collect 1 tube every 20 min with a collection volume of 26 µL. The control group was PD mice. High performance liquid-fluorescence chromatography was used to detect metabolite content in mouse brain dialysate to observe the effect of drugs on neurotransmitters in dialysate (Table S1).

RNA sequencing

Midbrain tissue samples were obtained from mice, RNA was extracted by the Trizol method, and RNA quality was detected using NanoDrop2000. Enrich eukaryotes’ mRNA with magnetic beads containing Oligo(dT), followed by the addition of fragmentation buffe to randomly interrupt the mRNA. Using mRNA as a template, the first strand of cDNA was synthesized with random primers. Buffer, dNTPs, and DNA polymerase I were then added to synthesize the second strand of cDNA. Double-stranded cDNA was purified using AMPure XP beads. The purified double-stranded cDNA was then end-repaired, A-tailed, and ligated sequencing linkers, followed by fragment size selection with AMPure XP beads. Finally, PCR enrichment was performed to obtain the final cDNA library. The library was tested for quality, and machine sequencing was carried out only after the test results meet the requirements. The raw image data files obtained by high-throughput sequencing were converted into the original sequencing sequence by CASAVA Base Calling Sequenced Reads. Results were stored in the FASTQ file format, which contains sequence information for sequencing sequences (reads) and their corresponding sequencing quality information. Finally, HISAT2 software was used to sequence compare Clean Reads with the specified genome to obtain its position information on the reference genome.

Bioinformatics analysis

Use featureCounts software to calculate the FPKM value expressed in each sample for each factor. DESeq2 was used for differential expression analysis of genes, and the p-value obtained from the original hypothesis test was corrected. The default was to use padj < 0.05, |log2(fold change)|>1 as the criteria for screening differentially expressed genes. Gene ontology (GO) enrichment and pathway analysis were used to understand the physiological properties of this protein using Database for Annotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/tools.jsp), including cellular components (CC), molecular functions (MF), biological processes (BP). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to annotate gene pathways.

Statistical analysis

The data was mainly analyzed using GraphPad Prim 9.0 software. The t-test was used for comparisons between the two groups, and one-way ANOVA was used for three or more groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns, no significant.