Yunnan Baiyao-loaded multifunctional microneedle patches for fast hemostasis and cutaneous wound therapeutic | Journal of Nanobiotechnology

Supplies and animals Gelatin, methacrylic anhydride (MA), and 2-hydroxy-2-methylpropylenone (HMPP) had been obtained from Shanghai Aladin Co., Ltd., China. Bletilla polysaccharide (BSP) was bought from Bai chuan Bio-technology Co., Ltd, China. Carbomer 940 was acquired from Beijing Solaibao Know-how Co., Ltd, China. rRtEGF was purchased from Shanghai PrimeGene Bio-tech Co., Ltd, China. Yunnan Baiyao (BY) was bought from Yunnan Baiyao Group. Gelatin methacrylate (GelMA) was self-synthesized in our laboratory. Deionized water (DIW, 18.25 MΩ·cm^− 1, Millipore) was used by all experiments. Male Sprague-Dawley rats weighted 125–150 g had been offered by Important River Laboratory Animal Know-how Co., Ltd. Any animal experimental protocols had been authorized by the Animal Investigation Ethics Committee of Wenzhou Institute of College of Chinese language Academy of Sciences (No. WIUCAS22092203).

Synthesis of GelMA GelMA was synthesized in our laboratory as we beforehand printed [32]. Sometimes, the gelatin (10%, w/v) and Na₂CO₃ (5%, w/v) had been dissolved in deionized water (DIW) and stirred for two h. 5 mL of methacrylic anhydride was dropwise added into 200 mL of gelatin resolution inside 30 min and stirred for one more 2 h. Throughout the response course of, the pH worth of the combined resolution was adjusted by NaOH resolution (1 M) for sustaining 8–9. The resultant resolution was dialyzed in opposition to DIW earlier than lyophilization to acquire GelMA. The diploma of substitution was examined from ¹ H NMR spectra (QUANTUM-I-400 MHz, Q.One Devices, China).

Fabrication of (BY + EGF) @MNs GelMA (20%, g/mL), EGF (0.002%, g/mL) and HMPP (1%, g/mL) had been dissolved within the DIW to acquire the EGF-GelMA pre-gels, which 100 µL EGF-GelMA pre-gels had been used to fill the MN tip cavity within the polydimethylsiloxane (PDMS) mildew utilizing a vacuum pump. Extra resolution was eliminated (≥ 50 µL) after which cured underneath UV mild (365 nm, 10 W, 10 s). BSP (10% w/v), Carbomer 940 (0.6%, g/mL) and BY (0.8%, g/mL) had been dissolved within the DIW to acquire the BY-BSP pre-gels, which about 100 µL BY-BSP pre-gels had been used to fill MN base cavity within the PDMS mildew and subsequently frozen at –80 ℃. The (BY + EGF)@MNs had been lastly obtained after lyophilization.

Characterization The intense-field and fluorescence micrographs of (BY + EGF)@MNs had been noticed utilizing a stereomicroscope (Olympus BX51, Tokyo, Japan). The microscopic buildings of (BY + EGF)@MNs had been recorded by a scanning electron microscope (SU8010, Hitachi, Japan). The compression and tensile exams of (BY + EGF)@MNs had been carried out on a common mechanical testing machine (5944, Instron, USA).

Mechanical characterizations The puncture power of MN ideas was measured by a compression check. The MN patches with totally different GelMA concentrations had been laid on a platform, the place the MN ideas pointed to the descending stress sensor. The sensor regularly moved to the ideas at 2 mm/min, and the pressure started to be recorded as 0.01 N when touching the MN ideas. The force-displacement curves had been recorded, and the pressure at 500 nm for various MN ideas had been calculated. As well as, the (EGF + BY)@MNs had been inserted into the rat pores and skin and liver tissues, which had been stained with H&E to guage the puncture depth.

Tissue adhesion exams The adhesion power between the pores and skin tissues and MN bases was verified by a tensile check. The pig pores and skin tissues had been firstly mounted to the horizontal stage. MN bases with gradient Carbomer contents had been contacted with the pores and skin tissues underneath a pre-pressure of 5 N for 1 min. The upside stress sensor hooked up to the MN bases was stretched upwards at a pace of 20 mm/min. The stretching course of was terminated manually when the MN bases had been separated from the pores and skin tissues. The utmost tensile pressure could possibly be obtained from the force-displacement curves as an index to guage the adhesion capacity of the MN bases.

BSP dissolution and BY launch The discharge kinetics of BSP and BY had been assessed utilizing a UV absorption spectroscope (obtained by Agilen CARY5000, USA). BSP and BY had the very best absorbance at 280 nm and 260 nm. The BSP patches with totally different Carbomer concentrations had been positioned in 5 mL of PBS (PH 5.6). Subsequently, 0.5 mL of PBS leaching resolution was taken out each 1 min, after which an equal quantity of an identical PBS was added instantly. By measuring the absorbance at 280 nm, the BSP dissolution charge could possibly be calculated primarily based on the usual curve of BSP resolution. Equally, the discharge kinetics curves of BY had been obtained by treating BY-containing BSP movie (BY@FN) in PBS resolution underneath totally different pH circumstances (i.e., pH = 5.6, 6.5, and seven.4) with the identical manner.

GelMA Degradation and drug launch The degradation charge of GelMA MN ideas was evaluated by recording their weight reduction. We ready cube-shaped GelMA hydrogel samples (measurement: 16 mm× 16 mm× 1 mm) to keep away from interference of BSP-based MN bases. All samples had been immersed in 1 ml of PBS with collagenase II (2.6 U/mL), stored shaking at 37 °C. The PBS resolution was changed at common intervals after centrifugation, after which 1 mL of recent PBS was supplemented. The MNs had been dried utterly at 60 °C and their dry weights had been recorded at designed time pints. The degradation curve was plotted primarily based on the load loss. For drug launch check, BSA was used as a drug mannequin and be integrated into the identical cube-shaped GelMA hydrogel to keep away from interference of BSP-based MN bases. With the identical method as above, the absorbance of BSA launched within the leaching resolution had been detected utilizing UV spectroscopy at every time level. The BSA launch profiles had been obtained underneath totally different pH circumstances.

RBC and platelet adhesion assays Gauze (scientific cotton gauze), BSP@FN (BSP flat patches, fabricated by 100 µL pre-gels with 10% BSP and 0.6% carbomer (w/v) after freeze-drying), BY@FN (BY-loading BSP flat patches, fabricated by 100 µL BY-BSP pre-gels (g/mL) containing 0.8% BY, 10% BSP and 0.6% carbomer), and (EGF + BY)@MNs (MN patches MN patches, fabricated by 100 µL BY-BSP pre-gels (g/mL) containing 0.8% BY, 10% BSP and 0.6% carbomer and 50 µL EGF-GelMA pre-gels (g/mL) containing 0.002% EGF and 20% GelMA ) had been used as experimental teams. The adhesion experiments had been carried out as printed [33]. RBC suspension and platelet-rich plasma (PRP) had been separated from the citrated complete blood (CWB) by centrifugation (1500 rpm, 10 min).

For RBC adhesion assay, the RBCs suspensions (100 µL) had been paved on the MN patch floor. After 1 min, they had been washed to take away non-adherent RBCs by utilizing PBS resolution, after which soaked in DIW (4 mL) to launch hemoglobin. After 2 h, the supernatant (100 µL) was pipetted, and the OD worth was measured at 540 nm (OD _experimental). The OD worth of DIW (4 mL) with RBCs suspension (100 µL) and PBS (100 µL) was measured as optimistic worth (OD _optimistic) and unfavorable worth (OD _unfavorable), respectively. The speed of adhered RBCs was calculated by the next Eq. (1):

For platelet adhesion assay,100 µL of PRP was paved onto the pattern surfaces for 1 min. Non-adherent platelets had been eliminated with PBS, after which the samples had been soaked in 2 mL of Triton X-100 resolution to free the lactate dehydrogenase (LDH). The LDH contents within the supernatants had been decided utilizing a LDH package (Solarbio, China). The OD_{450 nm} worth of the Triton X-100 resolution containing 100 µL of PBS and PRP was used as unfavorable worth (OD _unfavorable) and optimistic worth (OD _optimistic), respectively. The ratio of adhered platelets was calculated by the next Eq. (2):

As well as, the adhered RBCs and platelets had been noticed by SEM. The SEM samples had been ready by being incubated with 100 µL RBC or PRP for 1 min, washed with PBS, mounted in 3% glutaraldehyde for 12 h, dehydrated in gradient ethanol options, dried with a supercritical dryer, after which sprayed with gold by a high-vacuum ion sputtering machine (Leica, EM ACE600).

Coagulation assessments The blood clotting check of (EGF + BY)@MNs was carried out in accordance with earlier research [34]. Regular (with out therapy), BY (BY powers, 1 mg), BY@FN (BY-loading BSP flat patches, fabricated by 100 µL of BY-BSP pre-gels containing 0.8% BY, 10% BSP and 0.6% carbomer), and (EGF + BY)@MNs (MN patches with a BY-loading BSP base and EGF-loading GelMA ideas, fabricated by 100 µL of BY-BSP pre-gels and 50 µL of EGF-GelMA pre-gels (g/mL) containing 0.002% EGF and 20% GelMA) had been floor and combined with the 1 mL of serum from CWB. The clotting instances of various teams had been recorded at 37 ℃ by the PT/APTT assay reagent kits (Beijing ZONCI Know-how Improvement Co., Ltd, China).

Platelet activation assessments P-selectin (CD 62p) immunofluorescence staining was carried out to guage the activation impact of (EGF + BY)@MNs. The samples preparation in several teams had been as identical as RBC and platelet adhesion assays, which had been fabricated with co-incubated with PRP for 10 min, horizontally positioned on glass slides, after which mounted in glutaraldehyde for 12 h. The staining was carried out sequentially by CD 62p monoclonal antibody, after which examined utilizing a fluorescence microscope.

Hemocompatibility assessments Hemocompatibility of (EGF + BY)@MNs was assessed by remark and quantification of hemoglobin launched from the RBCs incubated with totally different samples. RBC suspensions had been acquired from CWB by centrifugation. 100 µL of RBCs suspension was combined with 900 µL of leaching resolution for various samples, which was centrifuged after incubation for 1 h. This hemolysis course of was photographed utilizing a digital digital camera. The OD values in several experimental teams had been recorded as OD _experimental at 550 nm. 100 µL RBCs suspension was dropped into DIW and used as optimistic management (OD _optimistic), whereas PBS was used as unfavorable management (OD _unfavorable). The hemolysis charge was obtained by following Eq. (3):

Cytocompatibility assessments 0 and 1 mg/ml of leaching medium of (BY + EGF)@MNs had been used as management and experimental group, respectively. NH3T3 cells had been added in a 96-well plate (800/effectively) and cultivated by totally different options. On days 1, 2, and three, the OD worth was detected at 450 nm after incubating with CCK-8 resolution (10% v/v, Beyotime Biotechnology) for 4 h. The NH3T3 cells had been stained with a reside/lifeless staining package for morphology remark.

Hemostasis and liver wound therapeutic in vivo In all animal experiments, SD rats had been divided into 7 teams: Ctrl (with out therapy), BY (handled with BY powders, 1 mg), BY@FN (handled with BY-loading BSP flat patches, fabricated by 100 µL of BY-BSP pre-gels containing 0.8% BY, 10% BSP and 0.6% carbomer), EGF (handled with 10 µL of 0.01% g/mL EGF resolution), EGF@FN (handled with EGF-loading GelMA flat patches, fabricated by 50 µL of EGF-GelMA pre-gels containing 0.002% EGF and 20% GelMA), (EGF + BY)@FN (handled with EGF-loading GelMA and BY-loading BSP flat patches, fabricated by 50 µL of EGF-GelMA pre-gels and 100 µL of BY-BSP pre-gels), (EGF + BY)@MNs (handled with MN patches with EGF-loading GelMA ideas and a BY-loading BSP base, fabricated by 50 µL of EGF-GelMA pre-gels and 100 µL of BY-BSP pre-gels). The drug or patch in every group had been administered solely as soon as on day 0. Moreover, earlier than traumatic procedures, all animals had been anesthetized by injecting atropine sulfate (0.01 ml/100 g, 0.4 mg/mL, Shanghai full woo Biotechnology Co., Ltd., China) into muscular and Zoletil®50 (0.15 ml/100 g, 50 mg/mL, Virbac Co., Ltd., France.) into stomach cavity.

The in vivo hemostatic and liver regenerative capacities of the (EGF + BY)@MNs had been evaluated utilizing a liver puncture hemorrhage mannequin in SD rats. The rat stomach was opened after anesthetization. The livers had been laid onto a pre-weighed sterile filter paper. Subsequent, a “cross” wound with a size of 5 mm and a depth about 3 mm was made for hepatic hemorrhage. The liver wounds had been lined with totally different samples, and the hemostasis course of was recorded. The hemostasis time was measured by a timer. The blood loss was decided by weighting the filter papers. The reside tissues in several teams had been harvested after 28 days. H&E staining (Solarbio, Beijing), PAS staining (Beyotime, Shanghai) and IL-6 immunohistochemical staining (Servicebio, Wuhan) had been performed for histological evaluation.

In vivo tissue restore analysis A full-thickness cutaneous wound mode was established in rats to exhibit the tissue restore impact of (EGF + BY)@MNs. The cutaneous tissues on the again of anesthetized rats had been excised to kind a circle with a radius of 5 mm and handled with other ways in varied teams. The injuries had been divided into 7 teams: Ctrl (with out therapy), BY (handled with BY powders, 1 mg), BY@FN (handled with BY-loading BSP flat patches, fabricated by 100 µL of BY-BSP pre-gels (g/mL) containing 0.8% BY, 10% BSP and 0.6% carbomer), EGF (handled with 10 µL of 0.01% EGF resolution), EGF@FN (handled with EGF-loading GelMA flat patches, fabricated by 50 µL of EGF-GelMA pre-gels containing 0.002% EGF and 20% GelMA), (EGF + BY)@FN (handled with EGF-loading GelMA and BY-loading BSP flat patches, fabricated by 50 µL of EGF-GelMA pre-gels and 100 µL of BY-BSP pre-gels), (EGF + BY)@MNs ( handled with MN patches with EGF-loading GelMA ideas and a BY-loading BSP base, fabricated by 50 µL of EGF-GelMA pre-gels and 100 µL of BY-BSP pre-gels). Subsequently, all wounds had been lined with medical breathable tapes to keep away from undesirable scratches. For monitoring the wound therapeutic course of, the injuries had been photographed on days 0, 2, 6, and 10. The wound dressings weren’t renewed on the designed intervals, which could possibly be helpful for monitoring the in vivo degradation of (EGF + BY)@MNs. Regenerated pores and skin tissues had been collected from the sacrificed rats on day 10. Microanatomy evaluation was performed by typical H&E/Masson staining, and immunofluorescence staining of CD31, VEGF, α-SMA and VIM.

Statistical evaluation All quantitative evaluation had been performed as means with normal deviations (n ≥ 3). Variations of two teams and a number of teams had been analyzed by unpaired Scholar’s t-tests and one-way analysis-of-variance (ANOVA), respectively. Vital variations had been thought of when *p < 0.05, **p < 0.01, and ***p < 0.001, and ‘ns’ indicated no vital distinction.