Materials
Gelatin (240 g Bloom), 5-aminosalicylic acid (5-ASA), fluorescein isothiocyanate and, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), were purchased from Aladdin (Shanghai, China). Roflumilast, glutaraldehyde and manganese dichloride were obtained from Macklin (Shanghai, China). FITC-Dextran (average molecular weight 4kD, FD4) was purchased from Sigma-Aldrich (FD4-1G). Beta actin (β-actin) antibody (20536-1-AP), Occludin antibody (27260-1-AP), F4/80 antibody (28463-1-AP) and CoraLite594-conjugated antibody (SA00013-4) were bought from Proteintech (Wuhan, China). Phospho-CREB (Ser133) (87G3) Rabbit mAb (9198S) was obtained from Cell Signaling Technology (Shanghai, China). TNF-α ELISA kit (1217202) was bought from DAKEWE (Shenzhen, China). BCA protein kit (P0012) was purchased from Beyotime (Shanghai, China). cAMP-Glo™ Assay (V1501) was purchased from Promega. Male mice (C57BL/6J) were obtained from Comparative Medicine Centre of Yangzhou University. Other chemical reagents and kits were purchased from commercial sources.
Extraction of MVs
MVs were prepared according to the previous research with some modifications. EcN 1917 was cultured in Luria–Bertani (LB) medium for 24 h at 37 °C. Then, the bacteria were collected by centrifugation at 4000×g for 15 min and homogenized via powerful sonication (3 kW) to gain MVs. Next, MVs solution was purified by centrifugation at 1500×g for 15 min and ultracentrifugation at 150,000×g for 2 h. The sediment was dispersed in deionized water by ultrasound and the concentration of MVs was determined by BCA kit. In addition, dynamic light scattering diameter analysis was performed (Figure S1).
Preparation of gelatin nanoparticles
Gelatin nanoparticles were obtained by two-method desolvation. Namely, 0.2 g gelatin was dissolved in 20 mL of deionized water at 40 °C and then equivalent volume of 95% ethanol (v/v) was poured slowly into the solution. Next, supernatant was discarded and 60 mL 95% ethanol (v/v) was added into the gel dropwise at 40 °C. When it was finished, the solution was stirred vigorously and 0.2 mL of 25% glutaraldehyde (v/v) was added for sequent conjugation overnight. Next, the gelatin nanoparticles solution was dialyzed to remove remnant ethanol. Then, dynamic light scattering diameter analysis was performed (Figure S1). Lyophilization was performed for quantity of gelatin nanoparticles concentration.
Preparation of MnO2 nanoparticles
MnO2 was synthesized via the reaction between potassium permanganate and gelatin (mass ratio, 1:4) for overnight. Namely, 0.45 mL of 20 mg/mL KMnO4 was added into 3 mL of 12 mg/mL gelatin nanoparticles with vigorous stir. Ion was eliminated by dialysis in deionized water. Then, MnO2 was processed for XPS, dynamic light scattering diameter analysis.
Determination of MnO2
Formaldehyde oxime was prepared by mixing 10 g hydroxylamine hydrochloride with 5 mL of 35% formaldehyde and adding 95 mL H2O into 100 mL. 0.1 mL of MnO2 was dispersed in 1.9 mL HCl (1 mol/L) for 24 h reaction to generate Mn2+. Then, 0.1 mL of the above solution was added into 1.7 mL of NH4Cl–NH3 buffer (1 mol/L, pH 10–11). Next, 0.1 mL of formaldehyde oxime and 0.1 mL EDTA-4Na (1 mol/L) were mixed in the solution. The analysis was executed at 450 nm via ultraviolet–visible spectrophotometer (Figure S4).
Synthesis of MVs-based nanoparticles
100 μL of 5 mg/mL roflumilast (in ethanol) was added into 6 mL of 2 mg/mL MVs and the mixture was sonicated for 8 min (350 W). Then, free Rof was removed via low-speed centrifuge to obtain Rof@MVs (Rof, 40 μg/mL). To ensure all MnO2 was entrapped in Rof@MVs, different concentration ratios of Mn and MVs were explored and 1:20 was chosen (Figure S2). The synthesis of MnO2@MVs (MnO2, 80 μg/mL) was by the mix of 1.5 mL of 160 μg/mL MnO2 and 1.5 mL of 2 mg/mL of MVs with 4-min sonication (65 W). For the preparation of Rof&MnO2@MVs (Rof, 20 μg/mL; MnO2, 80 μg/mL), 1.5 mL of Rof@MVs (Rof, 40 μg/mL) and 1.5 mL of 160 μg/mL MnO2 were homogenized by 4-min sonication (65 W). Then, Rof&MnO2@MVs were observed via SEM. For further use, these nanoparticles were lyophilized. Rof&MnO2@MVs (Rof, 40 μg/mL; MnO2, 80 μg/mL) preparation was in the similar way. 200 μL of 5 mg/mL roflumilast (in ethanol) was added into 6 mL of 2 mg/mL MVs and the mixture was sonicated for 8 min (350 W). Then, free Rof was removed via low-speed centrifuge to obtain Rof@MVs (Rof, 80 μg/mL). 1.5 mL of Rof@MVs (Rof, 80 μg/mL) and 1.5 mL of 160 μg/mL MnO2 were homogenized by 4-min sonication (65 W). Then, these nanoparticles were processed for dynamic light scattering diameter analysis.
Preparation of FITC-labelled MVs-based nanoparticles
MVs reacted with FITC at a mass ratio of 1:50, in NaHCO3–Na2CO3 buffer (0.15 mol/L, pH 9.0) for 12 h. Next, the product was dialyzed 4 times to remove free FITC and restored at − 80 °C. The FITC-labelled nanoparticles, including Rof@MVs-FITC, MnO2@MVs-FITC, Rof&MnO2@MVs-FITC, shared the same method with normal nanoparticles mentioned above.
Determination of roflumilast in Rof@MVs
Rof@MVs were firstly dispersed in 3 times volume of acetonitrile for demulsification via ultrasound and then centrifuged to gather supernatant for HPLC analysis. The mobile phase was acetonitrile and Na2HPO4–NaH2PO4 buffer (0.01 mol/L, pH 4), with a volume ratio of 1:1, flowing at 1 mL/min. C18 column (4.6 × 250 mm, 5 μm, Agilent) served as the stationary phase and the signal was detected at 250 nm (Figure S3).
Ex vivo simulation of MVs-based nanoparticles elimination
H2O2 was dropped into Rof @MVs (2 μg/mL Rof), MnO2@MVs (8 μg/mL MnO2) and Rof&MnO2@MVs (2 μg/mL Rof, 8 μg/mL MnO2) until the correspondent concentration. Then the nanoparticle solution was detected via ultraviolet–visible spectrophotometer (Figure S5).
Stability of MVs-based nanoparticles in vitro
All nanoparticles were dispersed in simulated colon fluid (SCF, 0.05 mol/L KH2PO4, pH 7.4) at 37 °C and diameters were determined in various time intervals.
Sustained release of roflumilast in vitro
1 mL Rof@MVs (40 μg/mL Rof) was dialyzed in 20 mL SCF (2% v/v Tween 80) at 37 °C for 24 h. At the indicated time, 1 mL SCF was aspirated for HPLC analysis and another 1 mL fresh SCF was complemented. Similarly, 1 mL Rof&MnO2@MVs (40 μg/mL Rof, 160 μg/mL MnO2) was used for the same measurement.
Cell viability assay
RAW264.7 was seeded in 96-well plate at the density of 3 × 104 per well for overnight. Then, cells were incubated with different concentration of nanoparticles for 24 h. Next, cell viability was examined with CCK-8 kit according to the manufacture protocol (Figure S6).
Comparation of nanoparticle uptake between CT26 and RAW264.7
CT26 and Raw264.7 were seeded in 6-well plate at the density of 106 per well for overnight, respectively. Then, cells were rinsed with 1× PBS and replenished with fresh medium, containing 1 μg/mL LPS. Next, Rof@MVs-FITC (Rof, 1.25 μg/mL), MnO2@MVs-FITC (MnO2, 5 μg/mL), Rof&MnO2@MVs-FITC (Rof, 1.25 μg/mL; MnO2, 5 μg/mL) and MVs-FITC (62.5 μg/mL) were added into the medium for 30-min incubation. After that, the medium was abandoned, and cells were rinsed three times and collected with 1× PBS for flow cytometry (BD FACS Callibur).
Validation of the macrophage-target of MVs
Firstly, FITC-labelled MnO2 (MnO2-FITC) was obtained by the addition of 1 mg FITC into 3 mL of 160 μg/mL MnO2 nanoparticles in NaHCO3–Na2CO3 Buffer (0.015 mol/L, pH 9.0) for overnight reaction. Then, the product was dialyzed in deionized water for 5 times to eliminate the residual FITC. The synthesis of MnO2-FITC@MVs was same with that of MnO2@MVs as mentioned before. Next, RAW264.7 were grown in 6-well plate at the density of 106 per well for overnight. Then, the medium was discarded, rinsed with 1× PBS and complemented with fresh medium, containing 1 μg/mL LPS. After that, RAW264.7 were incubated with MnO2-FITC (MnO2, 5 μg/mL), MnO2-FITC@MVs (MnO2, 5 μg/mL) and MVs (62.5 μg/mL) for 3 h. Lastly, cells were rinsed three times and collected with 1× PBS for flow cytometry (BD FACS Callibur).
Observation of nanoparticles in the macrophage
RAW264.7 was cultivated in the confocal dish at a density of 105 per dish overnight. Then, cells were rinsed with 1× PBS and replenished with fresh medium, containing 1 μg/mL LPS. After that, cells were incubated with Rof@MVs-FITC (Rof, 1.25 μg/mL), MnO2@MVs-FITC (MnO2, 5 μg/mL), Rof&MnO2@MVs-FITC (Rof, 1.25 μg/mL; MnO2, 5 μg/mL) and MVs-FITC (62.5 μg/mL) for 0.5 h. Next, the medium was discarded, and cells were rinsed three times and observed in confocal laser scanning microscope (CLSM, Olympus FV3000). The excitation wavelength was 488 nm and the emission wavelength was 525 nm.
The elimination of MnO2 in macrophage
RAW264.7 were seeded in 6-well plate at the density of 106 per well for overnight. Then, cells were rinsed with 1× PBS and stimulated with LPS (1 μg/mL in fresh medium). Next, DCFH-DA (5 μmol/L), Rof@MVs (Rof, 1.25 μg/mL), MnO2@MVs (MnO2, 5 μg/mL), Rof&MnO2@MVs (Rof, 1.25 μg/mL; MnO2, 5 μg/mL) and MVs (62.5 μg/mL) were synchronously added into the medium for 30-min incubation. After that, the medium was abandoned, and cells were rinsed three times and collected with 1× PBS for flow cytometry (BD FACS Callibur).
Influence of manganese in cytosolic cAMP
RAW264.7 were seeded in 96-well plate at the density of 1.5 × 104 per well for overnight. Then, cells were rinsed with 1× PBS and stimulated with LPS (1 μg/mL in fresh medium). Next, various formulations of manganese were added into the medium for 30-min incubation, including high dose MnO2@MVs (MnO2@MVs-H, 5 μg/mL MnO2), low dose MnO2@MVs (MnO2@MVs-L, 0.5 μg/mL MnO2), MnO2 (5 μg/mL MnO2), MnCl2 (7.25 μg/mL) and MVs (62.5 μg/mL). After that, intracellular cAMP was detected by cAMP-Glo™ Assay in accordance with manufacture’s instruction.
Modulation of nanoparticles in cytosolic cAMP
RAW264.7 were seeded in 96-well plate at the density of 1.5 × 104 per well for overnight. Then, cells were rinsed with 1× PBS and stimulated with LPS (1 μg/mL in fresh medium). Next, various nanoparticles were added into the medium for 30-min incubation, including Rof@MVs (Rof, 1.25 μg/mL), MnO2@MVs (MnO2, 5 μg/mL), Rof&MnO2@MVs (Rof, 1.25 μg/mL; MnO2, 5 μg/mL) and MVs (62.5 μg/mL). After that, cellular cAMP was detected by cAMP-Glo™ Assay (Promega) in accordance with manufacture’s instruction.
Quantification of TNF-α secreted from macrophage
RAW264.7 were seeded in 24-well plate at the density of 1.5 × 105 per well for overnight. Then, cells were rinsed with 1× PBS and stimulated with LPS (1 μg/mL in fresh medium). Next, various nanoparticles were added into the medium for 6 h incubation, including Rof@MVs (Rof, 1.25 μg/mL), Rof@MVs (Rof, 2.5 μg/mL), MnO2@MVs (MnO2, 5 μg/mL), Rof&MnO2@MVs (Rof, 1.25 μg/mL; MnO2, 5 μg/mL), Rof&MnO2@MVs (Rof, 2.5 μg/mL; MnO2, 5 μg/mL) and MVs (62.5 μg/mL). After that, the supernatant was measured by TNF-α ELISA Kit (DAKEWE) in accordance with manufacture’s instruction.
Protein analysis in Western Blot
RAW264.7 were seeded in 6-well plate at the density of 106 per well for overnight. Then, cells were rinsed with 1× PBS and stimulated with LPS (1 μg/mL in fresh medium). Next, various nanoparticles were added into the medium for 30-min incubation, including Rof@MVs (Rof, 1.25 μg/mL), MnO2@MVs (MnO2, 5 μg/mL), Rof&MnO2@MVs (Rof, 1.25 μg/mL; MnO2, 5 μg/mL) and MVs (62.5 μg/mL). After that, cells were rinsed three times and harvested. Then, cells were dispersed in RIPA Lysis Buffer with proteinase inhibitors by sonication. The debris was removed by centrifugation at 14,000×g, 5 min at 4 °C. The supernatant was collected for SDS-PAGE (10%) electrophoresis. The protein was detected via p-CREB antibody and β-actin antibody.
Establishment of DSS-induced colitis
All animal experiments were approved by Jinling Hospital (2021DZGKJDWLS-00143). Male C57BL/6 mice with 20–25 g, were purchased from Comparative Medicine Centre of Yangzhou University and housed in a 12 h light–dark cycle at 25 °C. Mice were raised 1 week for acclimation before random assignment. To construct murine colitis, mice were allocated randomly into groups and received 3% DSS in drinking water for 6 days. Healthy mice were supplied with ordinary drinking water during the experiment.
In-vivo distribution of MVs-based nanoparticles
Male C57BL/6 mice with 20–25 g received 3% DSS for 6 days after 7-day acclimation. Then, mice were given enema with saline, Rof@MVs-FITC (Rof, 1 mg/kg), MnO2@MVs-FITC (MnO2, 4 mg/kg) and Rof&MnO2@MVs-FITC (Rof, 1 mg/kg and MnO2, 4 mg/kg). Various organs were gathered at the indicated time for fluorescent imaging via in vivo imaging system (IVIS, Berthold LB983 NC100).
Prophylaxis of DSS-induced colitis
Before enema, mice were deprived of food for 12 h. Then, mice were anaesthetized with isoflurane and received enema on day 2, day 4 and day 6. The dose was Rof@MVs (Rof, 1 mg/kg), MnO2@MVs (MnO2, 4 mg/kg), Rof&MnO2@MVs (Rof, 1 mg/kg; MnO2, 4 mg/kg) and 5-ASA (1.25 mg/kg). Body mass was measured daily until sacrifice. On the day 9, mice were euthanized and colons were collected for length measurement.
Pathology analysis of colon
The distal colon was collected and fixed in Carnot’s solution for 24 h for the sequent parafilm embedding. The morphology of colon tissue was demonstrated by hematoxylin and eosin (H&E) stain. Goblet cells in the mucus were manifested via Alcian blue stain.
Detection of TNF-α in colon
The distal colon was collected and homogenized in the distal colon was collected (1 mmol/L PMSF). The debris was removed by centrifugation at 14,000×g, 5 min at 4 °C and the supernatant was collected. Then, the protein concentration was determined via BCA assay. After that, the supernatant was measured by TNF-α ELISA Kit (DAKEWE) in accordance with manufacture’s instruction.
Detection of intestinal permeability
FITC-Dextran (average molecular weight 4kD, FD4) was used for the permeability examination. On the day 9, mice were deprived of food and water for 4 h and given FD4 (0.4 mg/g) via intragastric administration. Next, the blood was gathered retro-orbitally 3 h later. The fluorescent intensity of FD4 in serum was measured with microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm).
In vivo immunofluorescence imaging of Occludin
As tight junction protein was depleted in UC, Occludin was analyzed by immunofluorescence to evaluate the effect of nanoparticles. The distal colon was fixed in 4% paraldehyde for 24 h and embedded in OCT for frozen section. Sections were firstly blocked with 5% BSA for 30 min in room temperature. Then, the colon tissue was stained with rabbit Occludin polyclonal antibody (Proteintech, 1:2000) for overnight at 4 °C. Next, the primary antibody was washed away with PBST and sections were stained with CoraLite594–conjugated goat anti-rabbit IgG(H+L) for 2 h in room temperature. The tissue was rinsed three times with PBST to remove free antibody and stained with DAPI for 15–20 min. The fluorescent images were acquired via CLSM (Olympus FV3000).
Macrophage-target of MVs-based nanoparticles in vivo
Male C57BL/6 mice with 20–25 g received 3% DSS for 6 days after 7-day acclimation. Then, mice were given enema with Rof&MnO2@MVs-FITC (Rof, 1 mg/kg and MnO2, 4 mg/kg). After 2 h, mice were sacrificed and the distal colons were collected and fixed in 4% paraldehyde for 24 h and embedded in OCT for frozen section. Sections were firstly blocked with 5% BSA for 30 min in room temperature. Then, the colon tissue was stained with rabbit F4/80 polyclonal antibody (Proteintech, 1:2000) for overnight at 4 °C. Next, the primary antibody was washed away with PBST and sections were stained with CoraLite594–conjugated goat anti-rabbit IgG(H+L) for 2 h in room temperature. The tissue was rinsed three times with PBST to remove free antibody and added with DAPI. The fluorescent images were acquired via CLSM (Olympus FV3000).
Biosafety of MVs-based nanoparticles
Normal male C57BL/6 mice with 20–25 g, were housed and treated. Enema was performed on the day 2, 4 and 6 with the same dose in the experiment mentioned above, namely Rof@MVs (Rof, 1 mg/kg), MnO2@MVs (MnO2, 4 mg/kg), Rof&MnO2@MVs (Rof, 1 mg/kg; MnO2, 4 mg/kg) and 5-ASA (1.25 mg/kg). Everyday body mass weight was monitored and euthanasia was executed on day 9. Serum from each group was collected for further hepatocyte injury and renal toxicity examination. Additionally, heart, liver, spleen, colon, lungs and kidneys were obtained for H&E staining to evaluate organ injury.
Microbiome analysis of colon
After mice were sacrificed, feces were collected for 16s rDNA sequencing in LC-Bio Technologies (Hangzhou) Co., Ltd. Briefly, total DNA was extracted with cetyltrimethylammonium bromide (CTAB). Then, the sequence was amplified in polymerase chain reaction (PCR). The PCR products were purified by AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified by Qubit (Invitrogen, USA). The amplicon pools were prepared for sequencing and the size and quantity of the amplicon library were assessed on Agilent 2100 Bioanalyzer (Agilent, USA) and with the Library Quantification Kit for Illumina (KapaBiosciences, Woburn, MA, USA), respectively. The libraries were sequenced on NovaSeq PE250 platform. The Shannon, Simpson, and Chao1 indices were calculated to assess the alpha diversity of each sample. PCA were used to assess beta diversity. The 30 most abundant communities at the genus level are shown by visualization methods, for example stack column.
Statistical analysis
Graph Pad Prism 9.0 and Origin 9.0 were used for data statistics and statistical significance calculation. Data were presented as mean ± SD. Statistical analysis was performed via two-tail Student’s t test or one-way ANOVA multiple comparisons tests and Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
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