Lyophilized apoptotic vesicle-encapsulated adhesive hydrogel sponge as a fast hemostat for traumatic hemorrhage in coagulopathy | Journal of Nanobiotechnology

Animals

Male Sprague–Dawley (SD) rats (7–8 weeks, 250–300 g) and male C57BL/6J mice (7–8 weeks) had been obtained from the Experimental Animal Middle of Solar Yat-sen College East Campus (Guangzhou, China), maintained in pathogen-free amenities. Male New Zealand rabbits (4–5 months, 2–2.5 kg) had been obtained from Xinhua laboratory animal Middle (Guangzhou, China), and housed within the typical situation. All of the animals had been stored on commonplace 12 h gentle–darkish cycles and obtained water and meals at libitum. All experiments had been in accordance with the ethics committees of Solar Yat-sen College (SYSU-IACUC-2022001534 and 2022002535). Animals had been anesthetized by inhalation of isoflurane (1–2% isoflurane in oxygen, RWD, China) utilizing a small animal anesthesia machine (RWD, China). Anesthesia was maintained by intramuscular administration of Zoletil®50 (50 mg kg⁻¹, VIRBAC, France) and Xylazine Hydrochloride (50 mg kg⁻¹, Sheng Da, China), in accordance with the producer’s directions. Heparinized rat and rabbit fashions had been achieved by intravenous heparin resolution (Stemcell, Canada) administration (500 IU kg⁻¹) [63].

Antibodies and reagents

All antibodies, chemical compounds, and different reagents used on this research are listed in Extra file 1: Desk S1.

Isolation, tradition, and characterization of UCMSCs

As in our earlier research [66], human cords had been obtained from full-term cesarean part surgical procedure, with the knowledgeable consent of those donors. The cells used on this research had been authorised by the Medical Ethics Committee of the Hospital of Stomatology, Solar Yat-Sen College (KQEC-2021-59-01). After rinsed and eliminated vessels, the cords had been minimize into small items, adopted by digestion with collagenase kind I (2 mg ml⁻¹, Worthington Biochemical, USA) and dispase II (4 mg ml⁻¹, Roche Diagnostics, Germany) for 1 h at 37 °C. After that, the single-cell suspension was acquired by passing the cells by way of a 70 µm strainer (BD Biosciences, USA). All nucleated cells had been seeded onto dishes (100 mm, Corning, USA) and cultured in alpha-Minimal Important Medium (α-MEM, Invitrogen, USA) supplemented with 15% fetal bovine serum (FBS, Gibco, USA), 2 mM l-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA) at 37 °C in 5% CO₂. The medium of major UCMSCs was changed each 3 days. UCMSCs on the eighth-tenth passages had been used for additional experiments.

The UCMSCs had been characterised by move cytometric evaluation. Briefly, UCMSCs had been harvested and suspended in Stain buffer (BD Pharmingen™, USA) (5 × 10⁵ cells mL⁻¹), incubated with PE-conjugated anti-human CD29 (1:100, BD Biosciences, USA), PE-conjugated anti-human CD44 (1:100, Biolegend, USA), PE-conjugated anti-human CD90 (1:100, BD Biosciences, USA), FITC-conjugated anti-human CD34 (1:100, BD Biosciences, USA), and PE-conjugated anti-human CD45 (1:100, BD Biosciences, USA) for 30 min at 4 °C. Information had been analyzed by NovoExpress™ software program (NovoCyte, USA).

Induction of UCMSC apoptosis and isolation of apoVs

The UCMSCs with a confluence of 90–95% had been washed twice with 1 X phosphate buffered saline (PBS, Servicebio, China), added with α-MEM containing totally different concentrations of staurosporine (STS, Enzo Life Sciences, USA) (250, 500, or 750 nM), and incubated at 37 °C for 12 h in 5% CO₂, or added with α-MEM containing 500 nM STS and incubated for various time (4, 8, 12, or 16 h). ApoVs had been remoted from the medium of apoptotic MSCs by sequential centrifugation (800g for 10 min, 2000g for 10 min, and 16,000g for 30 min) at 4 °C as we reported beforehand [67]. Lastly, the pellet was washed as soon as with 0.22 μm-filtered PBS to purify apoVs.

Isolation of Exos

As in our earlier report [16], UCMSCs had been washed twice with PBS after which cultured in α-MEM for 48 h at 37 °C. Exos within the tradition supernatants had been remoted by sequential centrifugation (800g for 10 min, 2000g for 10 min, 16,000g for 30 min, and 120,000g for 120 min) at 4 °C.

Identification of UCMSC-derived apoVs

Cryo-electron microscopy (Cryo-EM)

The morphology of apoVs was noticed by Cryo-EM (Thermo Fisher, USA) as we beforehand described [16, 68]. Briefly, after pattern preparation, photos of apoVs had been collected at a dose charge of 40 e/pixel/s, uncovered for 1 s. The pixel dimension on the object scale was 1.584 Å (nominal magnification 92 Ok) and a couple of.557 Å, with the defocus set at about -3 μm.

Nanoparticle monitoring evaluation (NTA)

NTA was carried out by ZetaView® PMX120 (Particle Metrix, Germany) to investigate the whole numbers, dimension distribution, and membrane potentials of apoVs and Exos. ApoVs or Exos had been diluted by ultrapure water and measured at 11 totally different positions with a medium video high quality setting. The information was calculated by ZetaView® evaluation software program.

Circulate cytometric evaluation

The floor markers of apoVs had been recognized as in our earlier analysis [16]. Briefly, apoVs had been harvested and suspended in Stain buffer and incubated with PE-conjugated anti-human TF (1:50, Biolegend, USA), PE-conjugated anti-human Fas (1:50, Biolegend, USA), PE-conjugated anti-human CD9 (1:50, Biolegend, USA), PE-conjugated anti-human CD63 (1:50, Biolegend, USA), and PE-conjugated anti-human CD81 (1:50, Biolegend, USA), PE-conjugated anti-human CD29 (1:50), PE-conjugated anti-human CD44 (1:50), PE-conjugated anti-human CD90 (1:50), FITC-conjugated anti-human CD34 (1:50), and PE-conjugated anti-human CD45 (1:50) for 30 min at 4 °C. For PS detection, apoVs had been suspended in Annexin V Binding Buffer (BD Pharmingen™, USA) and stained with PE-Annexin V (1:50) at 4 °C for 20 min. The constructive charge of apoVs had been analyzed by NovoExpress™ software program.

Western blotting

Cells, apoVs, and Exos had been lysed by the RIPA Lysis Buffer System (Santa Cruz Biotechnology, USA) to extract protein. After quantification of protein focus by way of the Pierce™ BCA Protein Assay Equipment (Thermo Scientific, USA), similar quantities of protein samples had been loaded onto the NuPAGE™ 4%–12% Mini Protein Gel (Invitrogen, USA) and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes had been blocked by 5% bovine serum albumin (BSA, Sigma-Aldrich, USA) for 1 h at RT and incubated with major antibodies (1:200–1000 dilution) in a single day at 4 °C. After washing with TBS containing 0.1% Tween®20 (Amresco, USA) for 3 instances, the membranes had been incubated with species-related HRP-conjugated secondary antibodies (1:10,000 dilution) for 1 h at RT. The bands had been visualized by way of SuperSignal™ West Pico PLUS Chemiluminescent Substrate package (Thermo Scientific, USA) or SuperSignal™ West Femto Most Sensitivity Substrate package (Thermo Scientific, USA), after which evaluated by a ChemiDoc™ MP imaging system (BIO-RAD, USA).

Blood-related part assortment and preparation

Rat circulating complete blood (CWB) was collected from the inferior vena cava of wholesome male rats utilizing anticoagulant tubes (Kangvis, China) (3.8% sodium citrate: blood = 1: 9). The CWB was centrifuged (3000 rpm for 15 min at RT) to amass Rat packed purple blood cells (RBC). 5% hematocrit of RBC suspension was ready by diluting packed RBC (100 μL) with PBS (2 mL). Rat regular plasma (NP) was obtained from rat CWB by centrifugation (800g for 10 min at RT). Rat platelet-poor plasma (PPP) was obtained from NP after centrifugation (3000g for 10 min at 4 °C). Rat microparticle-depleted plasma (MDP) was obtained by filtering PPP (0.1 μm) as reported beforehand [40]. Human MDP was acquired from the blood of wholesome people utilizing the identical process as above and the donors had been knowledgeable consent.

Blood and plasma clotting experiment

The blood and plasma clotting experiment was carried out in accordance with a earlier report with some modifications [26]. Briefly, blood or plasma (40 μL) was incubated with apoVs (12.5 μL) within the move tube for 3 min at 37 °C. After that, CaCl₂ (0.2 M, 7 μL, Sigma-Aldrich, USA) was added and clotting time was recorded (inside 1200 s), with PBS or Exos as management.

TF and PS blocking

The purified apoVs had been resuspended in Annexin V binding buffer (2.4 × 10⁸ particles, 12.5 μL), incubated with Annexin V (1 µg mL⁻¹, AV, Sino organic, China) or Tissue issue pathway inhibitor (32 µg mL⁻¹, TFPI, Sino organic, China) for 30 min at 4 °C, then used for the plasma clotting assays.

Transfection of siRNA in UCMSCs

To abrogate TF expression in UCMSC-derived apoVs, the UCMSCs (50–70% confluent) had been transfected with siRNA-negative management (si-NC) or siRNA-TF (si-TF) (Santa Cruz Biotechnology, USA) utilizing the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, USA) and Opti-MEM Medium (Gibco, USA), following the producer’s instructions. Transfection effectivity was evaluated at 48 h post-transfection by way of western blotting. UCMSCs transfected with siRNA (120 nM) for 72 h had been used for apoV isolation.

Lyophilization of apoVs

In response to earlier analysis [17], apoVs (1.2 × 10¹⁰ particles, 100 μL) had been added to lyoprotectants (400 μL), containing 100 mM d-(+)-Trehalose dihydrate (Trehalose, Macklin, China) and 5% polyvinylpyrrolidone 40 (PVP 40, Coolaber, China). After being frozen in a single day at − 80 °C, the combination above was lyophilized utilizing a − 80 °C freeze drier (Shengwei, China) for 30 h to acquire lyophilized apoVs, which had been saved in sealed packing containers to be moisture-proof at − 80 °C or RT. Earlier than use, lyophilized apoVs in every tube had been rehydrated by including ultrapure water (500 μL).

Synthesis of apoV-encapsulated hydrogel sponge (apoV-HS)

Manufacture of phenylboronic acid grafted oxidized hyaluronic acid (PBA-HA)

Primarily based on earlier experiences [32, 56], hyaluronic acid sodium salt (1 g, HA, Yuanye, China) was dissolved in deionized water (DI, 100 mL) underneath stirring (200 rpm), after which 3-Aminobenzeneboronic acid (3-APBA, Macklin, China) and 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride (DMTMM, Macklin, China) had been added, respectively. The molar ratio of 3-APBA, DMTMM, and HA was 1:1:1. The combination was stirred (200 rpm for twenty-four h at RT), after which dialyzed (MWCO 3500 Da) utilizing DI for 3 days to eradicate the unreacted substance. The dialyzed resolution was lyophilized to acquire white spongy strong phenylboronic acid grafted hyaluronic acid utilizing a − 50 °C freeze drier (LABCONCO, USA). Phenylboronic acid grafted hyaluronic acid (1 g) was dissolved in DI (100 mL) adopted by sodium periodate (0.25 M, 10 mL, Macklin, China) added dropwise. After reacting in darkish for twenty-four h at RT, glycol (1 mL, Macklin, China) was added to terminate the response, after which transferred into the dialysis membrane (MWCO 3500 Da) to purify PBA-HA. After 3 days of dialysis utilizing DI, the purified PBA-HA was lastly lyophilized by a − 50 °C freeze drier and saved at 4 °C.

Synthesis of apoV-HS

PBA-HA (60 mg) was dissolved in PBS (500 μL) at RT for 1 h, and poly(vinyl alcohol) (40 mg, PVA, Aladdin, China) was dissolved in PBS (500 μL) at 95 °C for 1 h to organize 12% PBA-HA and eight% PVA, respectively. ApoVs (4 × 10¹⁰ particles, 50 μL) or PBS (50 μL) was added into lyoprotectants (200 μL), containing 200 mM trehalose and 10% PVP40, with dietary supplements of 12% PBA-HA (125 μL) and eight% PVA (125 μL) to formulate apoVs hydrogel or PBS hydrogel. The hydrogels had been frozen in a single day at − 80 °C after which lyophilized for 30 h utilizing a − 80 °C freeze drier to acquire apoV-HS or PBS hydrogel sponge (PBS-HS).

Water absorption and adhesive take a look at

As earlier research reported [26], apoV-HS (60 mg) was compressed and injected into DI (3 mL) to formulate hydrated apoV hydrogel. The hydrogel was weighted at set time factors. All samples had been replicated (n = 3) and the swelling ratio was calculated in accordance with following method:

Ms and Md represented the burden of pattern in swollen and dry states, respectively.

The apoV hydrogel was placed on a slide (CITOTEST, China). Afterwards, the center, liver, spleen, lung, kidney, or pores and skin (6 mm × 6 mm) of a mouse had been positioned on the hydrated apoV hydrogel, respectively. The slide was then inverted to look at the adhesive functionality of the hydrated apoV hydrogel.

Blood clot index (BCI)

BCI was calculated in accordance with a earlier report with some modifications [26]. Briefly, rat CWB (30 μL) and CaCl₂ (0.2 mol L⁻¹, 7 μL) was slowly dropped on the floor of the comparable quantity samples or plate (unfavorable management), respectively, and incubated at 37 °C for five min. PBS (5 mL) was fastidiously added with out perturbing the blood clot. After that, specimens had been shaken gently (120 rpm for five min at 37 °C). The absorption of the answer at 540 nm was recorded by Multimode Microplate Reader (BioTek SynergyH1, USA). Surgifoam® and Avitene™ had been chosen because the constructive management.

The BCI of various supplies was measured by the next method:

the place OD_pattern was the absorbance of the answer from the samples at 540 nm, and OD_{reference worth} was the absorbance of the answer from the unfavorable management at 540 nm.

ApoV launch profile of apoV-HS in vitro

As beforehand reported [22], apoVs, labeled by PKH26 Crimson Fluorescent Cell Linker package (Sigma-Aldrich, USA) following the producers’ instructions, had been used to synthesize PKH26-labeled apoV-HS After being immersed in PBS (1 mL) at 37 °C, the supernatant of PKH26-labeled apoV-HS was collected in predetermined time factors (1 min, 3 min, 5 min, 7 min, 10 min, 1 h, 3 h, 7 h, 10 h, and 24 h). The fluorescence depth of the supernatant was measured at 570 nm by Multimode Microplate Reader.

Scanning electron microscope

ApoV-HS and PBS-HS had been compressed and injected into DI (1 mL) in an Eppendorf tube. After 10 min, ApoV-HS and PBS-HS had been hydrated, frozen at − 80 °C in a single day, after which subjected to lyophilization for 30 h utilizing a − 80 °C freeze drier. The freeze-dried PBS-HS and apoV-HS had been sprayed with gold by Sputter Coater (MC1000, Hitachi, Japan) for electrical conduction, and noticed by Scanning electron microscope (SEM, Quanta200, Thermo Fisher, USA).

Biocompatibility analysis in vitro

PBS-HS or apoV-HS (60 mg) had been incubated in Excessive glucose Dulbecco’s Modified Eagle Medium (25 mL, DMEM, Biosharp, China) supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C for twenty-four h to organize the extracts, as in earlier report [63]. Cell viability was measured by contacting extract.

CCK-8 assay

The cell counting kit-8 (CCK-8, Dojindo, Japan) was used to evaluate cell proliferation in accordance with the producer’s protocol. Particularly, L929 cells (mouse fibroblasts) had been seeded (1 × 10⁴ per effectively) into 96-well plates (Corning, USA) and handled with the extracts or regular tradition medium for twenty-four h, 48 h, or 72 h, respectively. CCK-8 (10 μL) was added to every effectively and incubated for two h. The absorbance of the CCK-8 resolution was detected at 450 nm by Multimode Microplate Reader.

EdU proliferation assay

The cell proliferation was additional carried out by the kFluor488-EdU Cell Proliferation Detection Equipment (KeyGEN, China). Briefly, L929 cells (5 × 10⁴ per effectively) had been seeded into 24-well plates (Corning, USA), cultured with the extracts or regular tradition medium for twenty-four h, and subsequently incubated with EdU for two h. Cells had been fastened by 4% paraformaldehyde (PFA, Sigma-Aldrich, USA), then underwent staining in accordance with the producer’s directions, and mounted with DAPI (Abcam, USA). The cells had been imaged utilizing fluorescence microscopy (ZEISS, Axio Observer 5, Germany), and the variety of proliferating cells was calculated.

In vivo experiments

The PBS-HS and apoV-HS utilized in all animal experiments had been ready by aseptic strategies [63].

In vivo biocompatibility analysis

The rat subcutaneous implantation experiment (n = 6) was in accordance with a earlier research [63]. After anesthesia and hair removing of the again, a subcutaneous pocket was generated by dorsal pores and skin incisions (1 cm in size) and blunt dissection, and PBS-HS (60 mg) or apoV-HS (60 mg) was implanted into it, with no implantation as management. Three implants had been positioned in every rat, making certain no overlap between the subcutaneous pockets. Subsequent, the injuries had been closed by interrupted sutures (4–0, JZ, China) and saline (5 mL) was injected subcutaneously. After 2 weeks and 4 weeks, the rats had been euthanized by cervical dislocation underneath anesthesia to gather the implants and adjoining tissue. There have been nonetheless residues that may very well be visually distinguished. Subsequent, the samples had been fastened by 4% PFA for hematoxylin–eosin (HE) staining and noticed by microscope (ZEISS, Axio Observer 5, Germany). Infiltrating inflammatory cells had been detected by IF staining. Briefly, the sections had been blocked with 5% BSA for 1 h at RT, then incubated with major antibody CD3 (1:100) in a single day at 4 °C. Subsequent, the sections had been washed and stained with Alexa Fluor 561-conjugated goat anti-rabbit secondary antibody (1:200) for 1 h at RT, then washed and counterstained with DAPI.

Heparinized rat liver bleeding mannequin

As beforehand reported [55], after injection of heparin resolution intravenously for 30 min, median laparotomy was carried out in rats, and the left anterior liver lobe was uncovered. Interstitial fluid surrounding the liver was wiped off with medical gauzes (Zeguan, China) to make sure the accuracy of blood loss. The biopsy punch (Kai, Japan) was used to create a cylindrical wound (8 mm in diameter, 5 mm in depth) within the left anterior lobe of the liver. The encompassing blood was wiped away to make sure correct measurement of blood loss, and apoV-HS was rapidly utilized on the defect website. For comparability, a comparable quantity of PBS-HS, industrial hemostats (Avitene™ and Surgifoam®), and no handled had been chosen as management. Every group contained 4 animals. The weighed filter paper was positioned underneath the liver to soak up blood. The bleeding time was recorded (inside 5 min), and the filter paper was weighed once more to calculate blood loss. After 4 weeks, all explants and adjoining hepatic tissue had been extracted, fastened by 4% PFA and embedded in paraffin. Paraffin sections (4 μm) had been ready for HE staining and noticed by microscope. IF staining was used to indicate CD3⁺ T cells within the liver.

Heparinized rat femoral artery/vein bleeding mannequin

As described beforehand [69], the left femoral artery and vein of the rats had been uncovered and minimize at 5 min post-heparin resolution injection. After 10 s free bleeding, blood across the bleeding level was wiped away, and apoV-HS (60 mg), PBS-HS (60 mg) or the comparable quantity of business hemostats (Avitene™ and Surgifoam®) had been utilized on the bleeding vessel instantly. In the meantime, pre-weighed gauzes and a weight (200 g) was positioned on the wound as fixed stress. The bleeding time (inside 20 min) and blood loss had been recorded and every group contained 4 rats.

Heparinized rabbit femoral artery bleeding mannequin

In response to a earlier research [55], the left femoral artery of rabbits was uncovered at 10 min post-heparin injection and surrounding interstitial fluid was eliminated by medical gauzes. Extreme arterial hemorrhage was executed by puncturing the femoral artery with a syringe needle (1.2 mm in diameter), with free bleeding for 10 s. Blood across the wound was rapidly wiped off to make sure an correct evaluation of blood loss. ApoV-HS (120 mg) was utilized to the bleeding artery instantly and lined with pre-weighed gauzes and a weight (200 g). The hemorrhage was checked each minute. No handled and a comparable quantity of Surgifoam® had been chosen as management teams, and every group contained 4 rabbits.

Statistical evaluation

Statistical and graph analyses had been carried out utilizing GraphPad Prism 9.0 (IBM, USA). For 2-group comparisons, significance was assessed by unpaired Pupil’s t take a look at. Variations amongst a number of teams had been analyzed by one-way ANOVA with Tukey’s submit hoc take a look at. Survival charge was analyzed utilizing log-rank Mantel-Cox take a look at for 2 and a number of group comparisons. All the info had been introduced as imply ± commonplace deviation (SD). ns, no important. *p < 0.05; **p < 0.01; ***p < 0.001.