Self‐Healing and Injectable Hydrogel for Matching Skin Flap Regeneration

Abstract The fabrication of highly biocompatible hydrogels with multiple unique healing abilities for the whole healing process, for example, multifunctional hydrogels with injectable, degradation, antibacterial, antihypoxic, and wound healing–promoting properties that match the dynamic healing process of skin flap regeneration, is currently a research challenge. Here, a multifunctional and dynamic coordinative polyethylene glycol (PEG) hydrogel with mangiferin liposomes (MF‐Lip@PEG) is developed for clinical applications through Ag–S coordination of four‐arm‐PEG‐SH and Ag+. Compared to MF‐PEG, MF‐Lip@PEG exhibits self‐healing properties, lower swelling percentages, and a longer endurance period. Moreover, the hydrogel exhibits excellent drug dispersibility and release characteristics for slow and persistent drug delivery. In vitro studies show that the hydrogel is biocompatible and nontoxic to cells, and exerts an outstanding neovascularization‐promoting effect. The MF‐Lip@PEG also exhibits a strong cytoprotective effect against hypoxia‐induced apoptosis through regulation of the Bax/Bcl‐2/caspase‐3 pathway. In a random skin flap animal model, the MF‐Lip@PEG is injectable and convenient to deliver into the skin flap, providing excellent anti‐inflammation, anti‐infection, and proneovascularization effects and significantly reducing the skin flap necrosis rate. In general, the MF‐Lip@PEG possesses outstanding multifunctionality for the dynamic healing process of skin flap regeneration.


Materials
Preparation of Mangiferin liposomes: thin-film dispersion method was utilized to prepare Mangiferin(MF)-loaded liposome(MF-Lip) as previously described. Specifically, materials containing phosphatidylcholine(PC)/ cholesterol(chol)/ MF (4:1:0.2 w/w) were dissolved in methanol with ultrasound to get uniform solution. Then the solution was transferred into rotary evaporator with the speed of 50 r/min at 40 C to get a faveolate film in the bottom of flask, under the vacuum condition, any trance of organic solution was eliminated. Further, the film was hydrated with preheated phosphate buffered saline (PBS) at 40 C with the speed of 50 r/min to get primary liposomes, then the solution was ultra-sonicated and extruded through the polycarbonate membrane filters with the pore size of 450 nm and 220 nm, respectively, to obtain homogeneous MF-Lip.

Characterization of Mangiferin liposomes: Physicochemical characterization of
Mangiferin liposomes: the size distribution, zeta potential and polydispersity indexes of MF-Lip were explored by Nano Series Zen 4003 Zeta Sizer. Further, the morphological characters of these nanoparticles were observed by transmission electron microscopy (TEM)  and Scanning electron microscope (SEM) (HitachiSU5000), separately.
To be more specific, the solution of MF-Lip was diluted to appropriate concentration, and then was stained with 2 % phosphotungstic acid to make the structure of liposomes was more easy to observe by TEM. Additionally, the liquid of MF-Lip was lyophilized under the protection of trehalose to get powder of MF-Lip used to explore the surface of these particles by SEM.
Drug release of Mangiferin liposomes in vitro: firstly, entrapment efficiency of MF-Lip was investigated as following description, 1 mL MF-Lip solution was put in the upper section of the ultrafiltration device (Millipore, Mw: 3500), and then was centrifugalized with the speed of 5000 r/min for 5 mins. When the process was complete, 1 mL fresh PBS was added into the upper part of the device and the centrifugalizing procedure was repeated to separate any unloaded MF. Subsequently, the concentration of MF in the bottomed solution was examined by high-performance liquid chromatography (HPLC) system with a UV detector (ShimadzuLC-2010A). The analysis was performed with an ODS column (100-5 C18 column, 250 × 4.6 mm, 5.0 m particle size, J&K Chemica LTD. Shanghai) at 35 C, and the mobile phases were consisted of acetonitrile and 0.1 % methanoic acid (170:830 v/v). Flow rate was 1.0 mL min -1 and the detection wavelength was set at 260 nm. Further, the MF-Lip was sealed in a dialysis bag (MW = 3500 Da) and then was immersed into PBS with (10 mM, pH 7.4) at 37 °C with the shake at 100 r/min. At setted time points, all of these release medium was replaced by fresh PBS, and the collected solution was tested by HPLC-UV to determine the concentration of MF. The test conditions were similar with abovementioned details. containing MF-Lip and AgNO 3 to fabricate the MF-Lip@PEG. In order to exhibit the injectable capability of MF-Lip@PEG, some rhodamine was added into the solution during the process of preparation to make hydrogels more apparent. Then these hydrogels were injected through 1 mL syringe (the diameter of needle is 0.5 mm), subsequently, these hydrogels were injected into water. In order to explore whether MF-Lip will make significant influences on the morphology of hydrogels, these hydrogels were lyophilized and then cut into slices, these samples were observed by SEM to explore the surface characters of hydrogels.

Preparation of hydrogels
Physicochemical characterization and drug release behavior of hydrogels: to explore the self-healing property of hydrogel, strain-dependent oscillatory measurements were firstly conducted on hydrogel to determine the critical strain value required to disrupt the gel network and transition to a solution state. Further, step-strain measurements were established to investigate the self-healing property of hydrogel, specifically, at the first 100 secs, hydrogel was subjected to low strain ( = 0.05 %) followed by high strain ( = 500 %) for 50 secs. When the high strain was discontinued then a low magnitude strain ( = 0.05%) was applied. Additionally, a hole was created in the center of hydrogel, 15 min later the appearance of hydrogel was recorded by a digital camera at macro to explore the process of self-healing.
MF-PEG, MF-Lip10@PEG, MF-Lip20@PEG and MF-Lip40@PEG hydrogel were freezedried to obtain the lyophilized samples. Subsequently, these hydrogels were weighted and immersed into DI water. At set time-point, these hydrogels were weighed and then datas were recorded, according to these datas, a curve was drawn to describe hydrogel's capability in water absorption. Those hydrogels for explore the degradation properties were soaked in DI water at 37 °C for 24 h to reach the equilibrium swelling state and then were weighed to record their initial masses. Further, hydrogels were kept in DI water at 37 °C with mild shaking, the remaining masses were regularly recorded at setted time points (days 1, 4, 8 and 12) to track the degradation kinetics.
The release behavior of these hydrogel were investigated by simulating drug release in vitro. To be more specific, these samples were immersed into PBS at 37 °C with the speed of 100 r/min in shaking bath. At setted time points, all release medium was replaced by fresh PBS, and these collected solution was analysed by HPLC-UV and related processing was similar as abovementioned details.

Macroscopic evaluation and histologic analysis:
The animals were anaesthetized and the surviving and necrotic areas of the flaps were photographed with a digital camera at day 7 of treatment, the necrosis of the flaps were determined by the color, gross appearance, tissue texture and the moorFLPI blood flow imager using the laser speckle contrast technique to capture real time blood flow images. The necrosis areas length were quantified as the percentage of the flap total length and the results were considered as percentage of skin flap necrosis. 1cm×1cm specimens were harvested from the necrosis and survival junction areas of the flaps for histological assessment. All tissue samples were fixed with 4% neutral formalin for 24 hours, then the specimens were embedded in paraffin and cross-sectioned into 5 µM slices. The slices were stained using heatoxylin-eosin for light microscopy.
Immunohistochemical analysis of CD31, CD68 and microvessel density detection: Immunohistochemiscal analysis was performed on 5 µM thick paraffin-embedded tissue sections. The sections were dewaxed and quenched the endogenous peroxidase activity with 3% hydrogen peroxide for 10 min, then blocked with the corresponding serum from a secondary antibody raised animal species for 1 h. The slices were incubated with the primary antibody (mouse anti-CD31 antibody,1:100; Abcam, USA; mouse anti-CD68 antibody,1:100; Abcam, USA), at 4 ˚C overnight, then added anti-rat secondary antibody at a dilution of 1:200 in PBS and incubated at room temperature for 30 min. After washed with PBS for several times, the signals on the tissue were revealed by incubating with DAB in PBS for 5~10 min, then hematoxylin counterstaining. The sections were observed under microscope.
Microvascular density was evaluated at 100× magnification, from the most vascularized area three areas were selected, then the microvessels were counted from those three areas at 200× magnification. The average count of the three areas was considered as the microvascular density.

Immunofluorescence staining of staphylococcus aureus and bacteria density detection:
Immunofluorescence analysis was performed on 5 µM thick paraffin-embedded tissue sections. The sections were dewaxed and quenched the endogenous peroxidase activity with 3% hydrogen peroxide for 10 min, then blocked with the blocking buffer for 1 hour. Slices were incubated with the primary antibody (mouse anti-staphylococcus aureus antibody,1:100;