GelMA/PEGDA microneedles patch loaded with HUVECs-derived exosomes and Tazarotene promote diabetic wound therapeutic | Journal of Nanobiotechnology



GelMA preparation

GelMA was synthesized in response to a beforehand described protocol [29]. Briefly, 10 g of gelatin (Sigma-Aldrich) was added to phosphate-buffered saline (PBS) to acquire 10wt% resolution beneath the 50 ℃. Then, 8 ml of methacrylic anhydride (Aladdin, Shanghai, China) was steadily added to the answer with stirring for six h at 50 °C. Lastly, 500 ml of heat PBS was added to finish the response. Dialysis utilizing a 13,000 molecular weight dialysis bag for 3 days to take away unreacted small molecules and freeze-dried to gather the product for future use.

β-CD-AOI2 preparation

β-CD-AOI2 was synthesized in response to a beforehand described protocol [30]. A easy nucleophilic response of ethyl isocyanate acrylate (AOI) (Aladdin, Shanghai, China) with cyclodextrin (Sinopharm Chemical Reagent Beijing Co. Ltd, China) gave cyclodextrin with double bond. 5 g cyclodextrin was dissolved in 50 ml DMF (Sinopharm Chemical Reagent Beijing Co. Ltd, China) containing 20 μl tin (II) 2-ethylhexanoate (Aladdin, Shanghai, China), and three ml AOI was slowly added dropwise to the answer to react for six h. The entire system was carried out in N2 ambiance. The response product was recrystallized with acetone and dried beneath vacuum to acquire the ultimate product for subsequent experiments.

Characterization of hydrogel

The 1H NMR spectra of the GelMA and β-CD-AOI2 have been collected from 0.7 ml pattern (10 mg ml−1) dissolved in D2O by NMR system (Bruker AVIII500 M, Switzerland). As well as, with the intention to characterize the structural traits of PEGDA (Aladdin, Shanghai, China) and GelMA, Fourier rework infrared spectroscopy (FT-IR) (Therno Nicolet, Nexus/Nexus, USA) was used. FT-IR spectra have been collected in transmission mode from 4000 cm−1 to 400 cm−1.

The mechanical properties of hydrogels with completely different PEGDA contents (0, 0.5%, 1%, 1.5%, 2%) have been analyzed by a low-force mechanical testing system. Hydrogels with a diameter of 1 cm and a peak of 4 mm have been ready for testing mechanical properties. The utmost loading power was set to 50.0 N, and the downward pace of the power sensor was set to 1 mm/min. The variation curve of compression power with deformation is recorded. The microstructure of hydrogels was analyzed by scanning electron microscopy. Firstly, the traditional hydrogel was brittlely damaged after freeze-drying, and its cross-section was noticed and analyzed by scanning electron microscopy (SEM, JSM-4800, Japan Hitachi). On the similar time, we concentrated the precursor resolution within the vacuum surroundings, after which obtained the hydrogel by picture crosslinking, and at last carried out an identical SEM evaluation.

Preparation and characterization of GelMA/PEGDA MNs

The MNs have been fabricated in response to a vacuum methodology. Briefly, 1 g of GelMA was dissolved in 10 ml of PBS resolution at 50 °C till totally dissolved. 25 mg Photoinitiator lithium acylphosphinate salt (LAP 0.05%, g/ml) have been added to the GelMA resolution for MNs fabrication. 100 μl of the above resolution was poured over a polydimethylsiloxane (PDMS) mould containing an array of MNs cavities. The MN was positioned in a vacuum surroundings at 50 °C take away air bubbles on the backside of the MN mould whereas sustaining the dissolved state of GelMA. After centrifugation, the answer was uncovered to UV mild for gel. Lastly, the MN arrays have been indifferent from the PDMS mould after holding the mould in the dead of night for twenty-four h to dry the GelMA/PEGDA MNs at room temperature.

To arrange a GelMA/PEGDA MN loaded with tazarotene (Shanghai, China) and exos, an acceptable quantity of tazarotene (1 mg/10 ml) was dissolved in a precursor resolution containing PEGDA, β-CD-AOI2 and GelMA. Then, after the dissolution of tazarotene was full, the exos (100ug/ml) have been added and combined evenly, and 500 ul of matrix resolution was crammed into PDMS mould for MN preparation.

The ready MNs have been put into 50 ml PBS resolution for degradation experiment. The MNs have been taken out at completely different time factors, wiped with filter paper, and dried after freezing. The MNs have been characterised by scanning electron microscope (SEM, JSM-4800, Hitachi, Japan).

The drug launch check was additional carried out. Every MN pattern was immersed in 10 ml PBS (pH = 7.4) at 37 °C. At every decided time level (1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d), 2 ml of launch medium was added into 2 ml of methanol, after which ultrafiltrated for detection. The discharge medium extracted from the unique launch system was changed with contemporary PBS of the identical quantity. The quantity of tazarotene launched from the combination was analyzed by microplate reader at 351 nm. In vitro, a GelMA/PEGDA@Tazarotene + exosomes (GelMA/PEGDA@T + exos) MNs patch was immersed with PBS at 37 °C. The variety of exos proteins have been detected utilizing Micro BCA Protein Assay Package (Thermo, 23,235, China) following the producer’s directions. In vivo, MNs patch loaded with PKH26 labeled HUVECs-exos and tazarotene was lined to the shaved again pores and skin of the diabetic C57BL mice, the pores and skin lined with MNs patch was harvested and fluorescence detection was then carried out on day 2, the unique MNs patch was continued to be lined to the opposite space of the pores and skin, the fluorescence detection was then carried out on day 4, and so forth, the identical therapy was carried out on day 6 and eight.

Cell isolation and tradition

Human immortalized keratinocytes (HaCAT) (#GDC106, CCTCC) and human umbilical vein endothelial cells (HUVECs) (#GDC166, CCTCC) have been obtained from the China Heart for Sort Tradition Assortment (CCTCC, Wuhan, China) and cultured following the producer’s directions. HaCAT and HUVECs have been cultured in Dulbecco’s modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). Cells have been cultured in a humidified ambiance containing 5% CO2 at 37 °C. Human foreskin fibroblasts have been remoted utilizing beforehand described protocols [31].

HUVECs-exos isolation and identification

Exosomes have been remoted utilizing a earlier protocol [32]. When HUVECs reached 85–90% confluence, they have been grown in an exos-free tradition medium. The FBS (Serapro) used for culturing HUVECs was ultracentrifugated at 120,000 g in a single day to take away contained extracellular exos. The tradition medium was obtained and centrifuged at 1,000 g for 10 min to take away lifeless cells and 4,000 g for 25 min to eradicate cell particles. The supernatants have been then moved to Amicon®Extremely-15 Centrifugal Filter (Millipore), and centrifuged at 13,000 g for 1 h. Subsequent, the supernatants have been centrifuged at 120,000 g for 80 min and 130,000 g for 70 min at 4 °C to acquire exos. The exos have been washed as soon as with PBS to take away contaminating proteins and saved at −80 °C for the following experiences. The pellets have been resuspended in PBS and saved at −80 °C.

The qualification of HUVECs-exos was carried out by transmission electron microscope (SEM, JSM-4800, Hitachi, Japan). The scale distribution of HUVECs‐exos was measured by Nanoparticle monitoring evaluation (NTA; Beckman Coulter). The overall protein degree was quantified Pierce BCA Protein Assay Package (Aspen, China).

HUVECs-exos labeling and internalization assay

To find out uptake of the HUVECs-exos by HaCAT, fibroblasts and HUVECs, HUVECs-exos have been incubated with pink fluorescent dye (PKH26, Sigma, USA) for five min and the neutralize redundant dye was eliminated by 0.5% BSA/PBS. Then, the residual dye was eliminated by centrifugation once more to acquire the labeled HUVECs-exos. Cells have been seeded within the confocal dish, after which handled with completely different concentrations (0, 5, 10, 20 ug/ml) of labeled HUVECs-exos. The cells have been washed with PBS after incubation for twenty-four h, after which mounted with paraformaldehyde (4%) for 10 min. FITC phalloidin (Yeasen Biotech Co., Shanghai, China) and DAPI (Solarbio, Beijing, China) have been used to stain the cytoskeleton and nucleic respectively.

Biocompatibility of the GelMA/PEGDA hydrogel in vitro

HaCAT, HUVECs, and Human foreskin fibroblasts have been used to research the toxicity of the GelMA/PEGDA hydrogel in vitro. GelMA/PEGDA hydrogel was immersed in a whole medium containing DMEM (excessive glucose) with 10% FBS for various durations of time (0 h, 24 h, 48 h, 72 h). Then the extract liquid was collected for the biocompatibility testing. The LIVE/DEAD assay (Beyotime, Shanghai, China) was used to check the cell exercise.

CCK-8 assay

Cell viability was estimated by the CCK-8 assay (Dojindo, Shanghai, China). Roughly 1.2 × 104 cells have been seeded in 96-well plates with 100 μl extract liquid per properly for twenty-four h, with three parallel controls for every group. Subsequent, 10 μl of the working reagent was added to every properly, and incubated at 37 °C for two h.

Scratch assay

The scratch assay was carried out in a 12-well plate. When the cells reached roughly 95% confluency with the extract liquid (48 h) per properly, the scratch was made through the use of a 200 μl pipette tip. A section distinction microscope was used to visualise the photographs. The photographs have been analyzed with Picture J model 5.0.

In vitro tube formation assay

HUVECs (2.5 × 104 cells per properly) have been seeded with the extract liquid (48 h) per properly in 96-well tradition plates that had been coated with 70 μl Matrigel Basement Membrane Matrix (BD Biosciences, CA, USA). Tube formation was detected through the use of the microscope at 0 h, 2 h, and 6 h incubation. Outcomes are represented as imply ± SEM in three in- dependent experiments.

Diabetic wound therapeutic mannequin

All animal experimental procedures have been accepted by the Institutional Animal Care and Use Committee of Tongji Medical School, Huazhong College of Science and Expertise. Eighteen wholesome eight-week-old male C57BL mice, weighing 20–23 g, have been bought from the Experimental Animal Heart, Tongji Medical School, Huazhong College of Science and Expertise. Streptozotocin (STZ) was used to induce wholesome C57BL mice to develop diabetes by intraperitoneal injection. The blood glucose ranges larger than 16.7 mmoll−1 at the least 1 month have been outlined as diabetic mice mannequin. Then the mice randomized into 4 teams randomly (n = 7 per group): the management group, GelMA/PEGDA MNs patch group, GelMA/PEGDA@Tazarotene (GelMA/PEGDA@T) MNs patch group, GelMA/PEGDA@Tazarotene + exosomes (GelMA/PEGDA@T + exos) MNs patch group. The C57BL mice have been anesthetized by 0.3% phenobarbital sodium (0.1 ml/10 g) by means of intraperitoneal injection. A ten-mm full-thickness cutaneous wound was excised on the midline line of the mouse again. Silicone ring was sutured to the wound edge to stop wound contraction. The injuries have been lined with PBS, GelMA/PEGDA MNs patch, GelMA/PEGDA@T MNs patch and GelMA/PEGDA@T + exos MNs patch. Digital images have been taken at days 0, 5, 10 and 15. The wound space was measured utilizing the Picture J software program.

Histology and immunofluorescence staining

After 15 days, the pores and skin was reduce into 10 mm*12 mm strips. Beneath the mechanical testing machine, the 50 N sensor stretched the pores and skin at a pace of 1 mm/min. The wound space pores and skin was eliminated and stuck with 4% paraformaldehyde for 48 h. The samples have been embedded in paraffin after dehydration. A microtome was used to acquire 5 μm-thick serial sections, which have been stained with hematoxylin–eosin (H&E), Masson trichrome, and immunofluorescence evaluation. The sections of wound areas have been respectively incubated with main antibodies α-smooth muscle actin (Abcam, 1:200) and CD31 (Abcam, 1:200) in a single day at 4 °C. Then they have been incubated with the second antibody (Servicebio) for 1 h at room temperature. Pictures have been taken through the use of a fluorescence microscope after which analyzed by Picture J software program.

Statistical evaluation

Statistical evaluation of knowledge was carried out by the unpaired Scholar’s t-test and one-way ANOVA amongst a number of teams. P < 0.05 was thought-about important. Statistical evaluation was carried out with the GraphPad Prism v 5.0 software program. Knowledge symbolize the imply ± SD of three impartial experiments.



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