Optogenetic engineered umbilical twine MSC-derived exosomes for transforming of the immune microenvironment in diabetic wounds and the promotion of tissue restore | Journal of Nanobiotechnology


Animals

Male C57BLKS-Leprdb (db/db) mice (8 weeks previous) have been bought from GemPharmatech Co., Ltd. All mice have been housed in a particular pathogen-free space and have been maintained in a 12-h mild: 12-h darkish cycle with managed temperature (24 °C ± 1 °C) and relative humidity (50–60%). All of the mice had free entry to meals and water. All surgical procedures and strategies used within the examine have been authorized by the Ethics Committee of Zhejiang Provincial Folks’s Hospital (No. A20220015).

To determine the mannequin of continual diabetic pores and skin wounds, the mice have been anesthetized with 4% (vol/vol) isoflurane (RWD Life Science, USA) earlier than the operation. After the animals have been shaved, a round full-thickness wound of 1.5 cm in diameter was created on the again of every mouse. The wound web site was noticed each day and was photographed utilizing a digital digital camera on days 0, 7, 14, and 21. ImageJ software program was used to measure and calculate the wound dimensions.

After surgical procedure, UCMSCs-exo/eNOS or UCMSCs-exo (20 μg dissolved in 100 μL phosphate-buffered saline) or an equal quantity of phosphate-buffered saline (PBS) have been injected subcutaneously into the wound each different day. On the indicated day, pores and skin tissue surrounding the wound was collected for evaluation.

Tradition of fibroblasts and endothelial cells

Human vascular endothelial cells (HUVECs) and L929 mouse pores and skin fibroblasts have been bought from the Chinese language Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Organic Industries, USA) and 1% penicillin–streptomycin (Thermo Fisher Scientific, USA). Cells have been incubated at 37 °C with 5% CO2.

Tubule formation assay

To check capillary-like development exercise in HUVEC cells, the formation of vascular-like constructions was assessed utilizing human fibrin matrices (Corning, USA). Briefly, chilly Matrigel (250 µL per effectively) was pipetted into 24-well plates utilizing a pre-cooled pipette tip. This was adopted by the addition of 400 μL of a suspension of three,3-Dioctadecyloxacarbocyanine perchlorate (DiO)-stained HUVECs (7.5 × 105 cells/effectively, untreated or handled with UCMSCs-exo/eNOS or UCMSCs-exo) on the Matrigel, and incubated at 37 °C for 12 h. Tube-forming capability was assessed by observing the tubular constructions utilizing a fluorescence microscope (Olympus, Japan).

Reactive oxygen species (ROS) detection

Intracellular ROS manufacturing was decided by 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining (Beyotime, China). Reactive oxygen species (ROS) in cells can oxidize non-fluorescent DCFH to supply fluorescent DCF, and the fluorescence depth of DCF can thus mirror the ROS degree. Fibroblasts or endothelial cells have been seeded in 6-well plates at a density of two × 105 cells/effectively. After reaching 80% confluence, the cells have been incubated with UCMSCs-exo/eNOS or UCMSCs-exo for twenty-four h after which subjected to high-glucose (HG, 50 mM) or H2O2 (200 μM) stimulation. After the indicated instances, cells have been incubated with 10 μm DCFH-DA for 20 min at 37 °C in serum-free medium after which washed thrice with PBS. The probe-loaded cells have been noticed by confocal laser microscopy and the fluorescence depth was analyzed by ImageJ software program.

Mitochondrial membrane potential measurement (JC-1 staining)

The mitochondrial membrane potential was detected by JC-1 staining (Beyotime, China). JC-1 is a perfect fluorescence probe that’s broadly used for the willpower of the mitochondrial membrane potential. When the mitochondrial membrane potential is excessive, JC -1 accumulates within the mitochondrial matrix and emits purple fluorescence. At low mitochondrial membrane potentials, JC-1 produces inexperienced fluorescence within the presence of the monomer. Fibroblasts or endothelial cells have been cultured in 6-well plates at densities of two × 105 cells per effectively and incubated with UCMSCs-exo/eNOS or UCMSCs-exo for twenty-four h. When the cells reached 80% confluence, they have been handled with hydrogen peroxide (200 μM) for 12 h. The JC-1 working answer was ready in accordance with the producer’s directions and was added to the wells and incubated at 37° C for 20 min. After three washes with buffer, the modifications in fluorescence have been monitored by fluorescence microscopy and the ratio of purple to inexperienced fluorescence depth was analyzed utilizing ImageJ.

TUNEL

Apoptosis of endothelial cells handled with H2O2 (200 μM) for 12 h was assessed utilizing a fluorescent TUNEL detection package (Beyotime, China), in accordance with the producer’s directions. The cells have been fastened with 4% paraformaldehyde at room temperature for 20 min, adopted by permeabilization with 0.3% Triton X-100 for five min, and the addition of the TUNEL detection answer. The cells have been incubated at 37 °C for 60 min in the dead of night. The nuclei have been counterstained with DAPI. TUNEL-positive cells have been evaluated and counted below confocal microscopy.

Histological evaluation

The pores and skin of the wound tissue was collected on postoperative days 7, 14, and 21, fastened with 4% paraformaldehyde, and embedded in paraffin after dehydration. The paraffin-embedded tissues have been sliced into 5-μm-thick sections. Re-epithelialization and the diploma of collagen maturation have been noticed by hematoxylin and eosin (H&E) or Masson’s trichrome staining. Photographs have been examined below an optical microscope (Olympus, Japan).

For immunohistochemical staining, the paraffin sections have been rehydrated and incubated with the first antibody, adopted by incubation with the secondary antibody and the streptavidin biotin–peroxidase advanced. Lastly, the samples have been visualized by the chromogenic substrate diaminobenzidine (DAB). The stained sections have been noticed with an optical microscope.

For the evaluation of immunofluorescence, the paraffin sections have been rehydrated, blocked with 1.5% goat serum, and incubated with the first antibody in a single day at 4 °C. The sections have been then handled with Alexa Fluor 488 and Cy3-conjugated secondary antibodies, whereas the nuclei have been stained with DAPI. The sections have been examined and imaged utilizing a confocal microscope and the fluorescence space and depth have been evaluated by ImageJ software program.

Quantitative real-time PCR (qRT-PCR)

Complete RNA was extracted with TRIzol reagent and complementary DNA (cDNA) was obtained by the reverse transcription of 1 μg of whole RNA from every extracted pattern utilizing the PrimeScript RT reagent package (Takara Biotechnology, Japan). Subsequent, the SYBR Inexperienced detection reagent (Takara Biotechnology) was used for qRT-PCR evaluation in an Utilized Biosystems 7500 Actual-Time PCR System (Utilized Biosystems, USA). Beta-actin or GAPDH was used for normalization of the outcomes. The sequences of the primers used on this examine are supplied in Extra file 4: Desk S1.

Western blotting

Briefly, tissues have been homogenized and lysed on ice for 30 min in pre-chilled RIPA buffer containing a phosphatase inhibitor cocktail and PMSF. The lysates of exosomes or tissues have been diluted in a 1:5 ratio with protein loading buffer (5 ×) (ABclonal, China) and heated at 95 °C for five min. Protein extracts have been separated on 4‒20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels at 120 V and blotted at 220 mA onto polyvinylidene di-fluoride (PVDF) membranes (Merck Millipore, Germany) for 90 min. The membranes have been blocked with Blocking Buffer (Yoche, China) for 10 min at room temperature adopted by in a single day incubation at 4 °C with the first antibodies. The next day, the membranes have been incubated with horseradish peroxidase (HRP)-linked secondary antibodies (HuaBio, China) for two h at room temperature. Lastly, protein bands have been visualized utilizing an ECL substrate package (Bio-Rad, USA) and the expression ranges of the proteins have been quantified by ImageJ. All major antibodies have been from Cell Signaling Know-how (USA). All protein expression was normalized to β-actin or GAPDH.

Tradition and identification of human UCMSCs

Human umbilical cord-derived mesenchymal stem cells have been supplied by Weiwei Biomedical Know-how (China) and their identification was accomplished with the total assist of Weiwei Biomedical Know-how. The UCMSC cell line was cultured in MesenCult™ MSC Basal Medium containing MesenCult™ MSC Stimulatory Supplemen (STEMCELL Applied sciences, Canadian). UCMSC floor marker proteins have been evaluated by circulate cytometry (Agilent, USA); these included three constructive markers (CD90, CD105, and CD73), six adverse cocktails (CD45, CD34, CD14, CD11b, CD19, and HLA-DR), and the respective isotype controls. All antibodies have been obtained from BD Biosciences (San Jose, CA, USA). Osteogenesis, adipogenesis, and chondrogenesis of UCMSCs have been evaluated utilizing the MesenCult™ osteogenic, adipogenic, and chondrogenic differentiation package (STEMCELL Applied sciences). Differentiation properties have been recognized in accordance with the producer’s protocol.

UCMSCs-exo/eNOS isolation and identification

The recombinant sort 5 adenovirus expressing two fusion proteins, CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 was contracted for manufacturing to Shanghai Genechem Co., Ltd. To upregulate CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 for protein–protein interactions utilizing optogenetics, the adenovirus vectors carrying the fusion protein CIBN-EGFP-CD9 or eNOS-mCherry-CRY2 have been transfected into UCMSCs in accordance with the producer’s protocol.

Ultracentrifugation was used for the isolation and extraction of exosomes. UCMSCs overexpressing CIBN-EGFP-CD9 and eNOS-mCherry-CRY2 have been seeded into T175 flasks. After sooner or later, the medium was rigorously eliminated, and exosome-depleted medium was added.

Then, the cells have been uncovered to steady blue mild illumination from a 460: 465 nm mild board in a CO2 incubator. After 72 h, the cell tradition supernatant was harvested and centrifuged at 300g for 10 min and 2000g for 30 min to take away lifeless cells and mobile particles. After additional centrifugation at 10 000g for 30 min, the supernatant was filtered via a 0.22-µm filter (Merck-Millipore). The supernatant was then centrifuged twice at 100 000g for roughly 1.5 h every. The pellets have been resuspended in PBS and saved at −80 °C for additional experiments. The separation and purification of UCMSCs-exo was carried out utilizing the identical procedures.

To confirm the ultrastructure and form of the exosomes, the exosomes have been evaluated utilizing transmission electron microscopy (TEM). Dynamic mild scattering (DLS, Malvern Devices, UK) was used for figuring out the dimensions distribution and particle focus of the exosomes. The presence of particular exosomal floor markers (TSG101, CD9, CD63, CD81) was evaluated utilizing Western blotting.

Exosome labeling and uptake

Exosomes have been labeled with the Dil fluorescent labeling package (Yeasen, China). Dil (10 μm) was added to an exosome suspension and incubated at room temperature for 20 min in the dead of night. Exosomes have been collected by centrifugation at 100 000g for 90 min after which washed twice with PBS to take away any unbound dye. The Dil-labeled exosomes have been then incubated with the HUVECs for twenty-four h. The cells have been then fastened and the nuclei have been stained with Hoechst. Photographs have been obtained by confocal microscopy.

Lipid peroxidation willpower

Lipid peroxidation was assessed by measuring the purple byproduct of the response of malondialdehyde (MDA) with thiobarbituric acid (T BA). Cells have been handled with UCMSCs-exo/eNOS or UCMSCs-exo for twenty-four h and uncovered to excessive glucose (50 mM) for twenty-four h. Absorbances have been measured and commonplace curves have been established in accordance with the producer’s directions (Beyotime, China).

SOD willpower

The exercise of superoxide dismutase (SOD) was decided by the WST-8 technique. WST-8 can react with xanthine oxidase-catalyzed superoxide anion radicals to supply water-soluble formazan dye and as SOD can inhibit formazan dye manufacturing via dissimilation, the exercise of SOD may be measured by colorimetric evaluation. SOD actions have been measured within the cells after totally different remedies in accordance with the producer’s directions (Beyotime, China).

Complete glutathione assay

This was measured by the discount of oxidized glutathione (GSSG) to decreased glutathione (GSH) by the mitochondrial enzyme glutathione reductase. GSH reacts with the chromogenic substrate DTNB to supply yellow TNB and GSSG. The whole glutathione content material was calculated by adjusting the response system and measuring the quantity of yellow TNB shaped. Every group of cells was measured after 24 h of high-glucose stimulation, in accordance with the producer’s directions (Beyotime, China).

NO measurement

NO is definitely oxidized to NO2– and NO3– in vivo and NO3– is decreased to NO2– by nitrate reductase. Beneath acidic situations, NO2– types diazo compounds with diazosalt sulfonamide, which may be additional coupled to naphthyl vinyl diamine. The product had a attribute absorption peak at 550 nm, permitting calculation of the NO content material by measuring the absorption worth. Pores and skin tissue was collected and homogenized. In keeping with the producer’s directions, the Micro NO Content material Assay Equipment (Solarbio, China) was used for NO measurements.

Transwell migration assay

Cell migration was measured by the Transwell technique utilizing 24-well Transwell tradition plates (Corning, USA) with 8 μm pore-sized filters was used. Roughly 1 × 104 cells have been inoculated into the higher chamber and incubated with serum-free medium. Then, totally different remedies containing HG (50 mM), UCMSCS-exo (20 μg/ml), and UCMSCs-exo/eNOS (20 μg/ml) have been added to the decrease chamber. Cells have been cultured for twenty-four or 48 h in an incubator at 37 °C, after which the medium was eliminated, and cells have been washed thrice with PBS. Subsequently, cells have been fastened with 4% paraformaldehyde for 15 min after which stained with 0.1% crystal violet for a number of minutes. A cotton swab was used to take away cells from the highest floor of the filter. Migratory exercise was assessed by observing the stained cells below an optical microscope and their enumeration utilizing ImageJ software program.

Cell migration assays

Cells (2 × 105 cells per effectively with three replicates per group) have been seeded in 6-well plates and incubated at 37 °C. After the cells reached confluency, the monolayer was manually scratched with the tip of pipette, and the indifferent cells have been eliminated by washing with serum-free medium. The cells have been then cultured in medium supplemented with or with out HG (50 mM), UCMSCS-exo (20 μg/ml), and UCMSCs-exo/eNOS (20 μg/ml). All cells have been handled with Mitomycin-C for 1 h earlier than scratching to exclude the affect of cell proliferation on wound closure. The matched wound areas have been photographed at 0, 24, 48, and 72 h after wounding. The migration space (%) was calculated as (A0-An)/A0 × 100%, the place A0 represents the preliminary wound space and An represents the remaining space of the wound on the measured time level.

Proliferation assay

Briefly, cells (5 × 103 cells per effectively, six replicates per group) have been seeded into 96-well tradition plates and handled with UCMSCs-exo (20 μg/ml) or UCMSCs-exo/eNOS (20 μg/ml). The cell-free group served because the clean group. At 12, 24, 36, and 48 h, the Cell Counting Equipment-8 reagent (CCK-8, Yeasen, China; 20 μL per effectively) was added to the medium (100 μL per effectively). After incubation at 37 °C for two h, the absorbance of every effectively was measured at 450 nm by a microplate reader. The OD values have been used to evaluate cell proliferation.

Calcein/PI cell viability/cytotoxicity assay

The survival and demise of cells have been assessed utilizing Calcein-AM (Calcein-AM) and PI (propidium iodide) double-fluorescence staining (Beyotime, China). Briefly, roughly 2 × 105 HUVECs have been seeded in 6-well plates and incubated in full medium with or with out UCMSCs-exo/eNOS (20 μg/ml) and UCMSCs-exo (20 μg/ml) for twenty-four h earlier than stimulation with H2O2 for 12 h. The calcein AM/PI detection answer was added and incubated for 30 min at 37 °C in the dead of night in accordance with the producer’s directions. The nuclei have been stained with Hoechst. The cells have been visualized by confocal microscopy.

Circulate cytometry

The connected adipose tissue was eliminated earlier than harvesting the pores and skin wound tissue and the pores and skin samples have been sliced and digested with the Opti-MEM (Invitrogen) options containing collagenase sort I (0.5 mg/ml, BioFroxx, China), collagenase sort II (0.5 mg/ml, BioFroxx, China), collagenase sort IV (1 mg/ml, BioFroxx, China), hyaluronidase (1 mg/ml, BioFroxx, China), and deoxyribonuclease I (0.02 mg/ml, Biosharp, China) for 30 min on a shaker at 37 °C. The tissue was mechanically floor utilizing a Tissue Grinder with Pestle (YiXi Bio, China), washed with a whole medium containing 10% FBS, and the purple blood cells have been lysed.

Cell surfaces have been stained in the dead of night on ice for circulate cytometry evaluation. The cells have been stained with antibodies towards the floor antigens CD4 (MultiSciences, China), CD25 (MultiSciences, China), CD8 (Proteintech, China), CD69 (Proteintech), and CD103 (Proteintech). Cells have been fastened and permeabilized after which incubated with anti-CD16/32 (MultiSciences, China) to dam nonspecific binding. Staining of intracellular markers, such because the Treg marker FoxP3 (MultiSciences, China), have been carried out on ice. The cells have been then analyzed by circulate cytometry.

Statistical evaluation

All information are offered as means ± commonplace deviation (SD). Unbiased-sample t-tests have been used to match the means between two totally different teams. One-way evaluation of variance (ANOVA) was utilized for the analysis of the numerous distinction. A number of-group comparisons have been carried out utilizing one-way ANOVA. GraphPad Prism 8 software program was used for all statistical analyses. P-values < 0.05 have been thought-about statistically vital.

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