Benlysta-Loaded Sodium Alginate Hydrogel and Its Selective Functions in Promoting Skin Cell Growth and Inhibiting Inflammation.

Benlysta is a new drug approved by the US Food and Drug Administration (US FDA) in 2019 for the treatment of systemic lupus erythematosus. In this study, we loaded the benlysta in the traditional sodium alginate (SA) hydrogel to investigate the potential application of the drug-loaded hydrogel for skin dressing or hypodermic drug. Live/dead staining images and the CCK-8 results showed that the benlysta-loaded hydrogel could promote the growth of human epidermal cells (HaCat), fibroblasts (L929), and endothelial cells while inhibiting the aggregation of inflammatory cells (macrophages). In addition, the hydrogel degradation and drug release are slow and controllable, and the gel time of drug-loaded hydrogel can be adjusted by adding sodium alginate ratios according to the requirement. In summary, we prepared a time-dependent drug-loaded hydrogel for potential application in the treatment of skin injury that may be caused by other diseases.

Introduction

As an important organ of human body, skin plays an important role in protecting the body from invasion and stimulation (temperature, hardness, humidity, pain, etc.) from the outside world. Therefore, skin damage brings a lot of discomfort and inconvenience to the human body. Especially, severe skin damage caused by external organic matter (scratch from a sharp weapon, burn, frostbite, etc.) and major diseases (skin disease, diabetes, lupus erythematosus, etc.) is not easy to heal. These skin injuries take a long time to heal, and it is an orderly and complex process that includes coagulation, inflammation regression, tissue regeneration, epidermal repair, etc.[1,2] To solve these problems and accelerate wound healing, a series of hydrogel dressing have been developed.[3−10] These hydrogel dressings greatly improve the microbial environment of skin wounds, effectively inhibit bacterial infection, and make up for the deficiency of antibacterial ability of skin wounds caused by major diseases.[11−13] However, the repair of skin wounds needs to be considered in many aspects. In addition to antibacterial properties, the inhibition of inflammation (macrophage participation),[14] the repair of connective tissue (fibroblasts participation),[15] epidermis (epidermal cell participation), and capillaries (platelets, endothelial cells, and smooth muscle cell participation) should also be considered.[16,17] For deeper skin wounds, where it is not enough to use hydrogels as dressings, they can also be injected.[18] Because most of the current prescription drugs have multiple functions, it is a preferable choice to use hydrogel loaded with prescription drugs as biomaterials for skin injury treatment.

In the previous work, we have developed a series of injectable time-dependent sodium alginate (SA) hydrogels for stem cells and/or molecular loading.[19,20] The advantage of sodium alginate is that it has huge storage capacity, and sodium alginate hydrogels can be formed under mild conditions when the molecule is combined with two valence cations. The sodium alginate hydrogels have been widely applied in tissue engineering due to their excellent biocompatibility, low toxicity, and immune response.[21] Additionally, they are also used as carriers for an accurate delivery of drugs, cells, genes, or proteins to improve the therapeutic effect.[22,23] Therefore, sodium alginate hydrogels are preferable materials for treating skin damage. British pharmaceutical giant GSK recently (April 2019) announced that the US Food and Drug Administration (US FDA) has approved the lupus drug benlysta for children aged 5 years and above with systemic lupus erythematosus (SLE) through priority review. SLE is the most common type of lupus, which will lead to serious long-term organ damage.[24] In this contribution, we tried to load the new drug benlysta into sodium alginate hydrogels and systematically evaluated the effect of benlysta-loaded hydrogels on the growth of macrophages (inflammation evaluation), epidermal cells, fibroblasts, endothelial cells, etc. to obtain a more widely used biomaterial for the treatment of skin injury.

Results and Discussion

Material Characterization of Benlysta-Loaded Hydrogels

Gelation is the most important feature and advantage of hydrogels.[25]Figure shows that after the addition of two valence ions (calcium ions), the mixture of sodium alginate (SA) and benlysta rapidly gelled and adhered to the container wall, suggesting excellent gelation property of the benlysta-loaded SA hydrogel. At the skin wound, there are enough divalent ions, such as Ca2+, Mg2+, Fe2+, Zn2+, etc.,[26,27] which can promote the rapid formation of the gelation. Figure shows that the 2% SA ratio had a shorter gelation time (3 min) than the 1% SA ratio (7 min). As skin dressing, a shorter gelation time certainly has a better healing effect; however, as an injectable material, it needs to take more than 5 min to treat deeper skin wounds. Thus, this result indicated that the gelation time can be determined by regulating the SA ratios according to different treatment needs of the wounds. The water content of the normal skin tissue is in the rage of 25–70%; the deeper the tissue, the higher is the water content.[28] The lack of water content will lead to dry, rough, and chapped skin tissue, leading to a series of skin problems.[29]Figure displays that benlysta-loaded hydrogels with 1 and 2% SA ratios presented no significant difference: both samples had a water content that exceeded 95%, which can fully meet the needs of skin moisture content.

Figure 1

Pictorial diagram of the gelation of the benlysta-loaded SA hydrogel.

Figure 2

Gelation time of the benlysta-loaded hydrogels with 1 and 2% SA ratios (**p < 0.01, mean ± SD, n = 3).

Figure 3

Water content of the benlysta-loaded hydrogels with 1 and 2% SA ratios (mean ± SD, n = 3).

It has been reported that the exudation of inflammatory fluid and tissue fluid often delays the healing of skin wounds, so it is helpful to absorb excessive surface fluid for wound healing.[30] In skin dressing, the better swelling rate is beneficial to remove excess water from the wound. Figure shows that the swelling rate of the benlysta-loaded SA hydrogel rapidly increased within 24 h and then remained at 150%. The drug release of the hydrogel is often affected by the water content and swelling rate, and Figure shows that the benlysta-loaded SA hydrogel released about 50% drug in 72 h; the initial release of drugs in high dose is conducive to rapid hemostasis, sterilization, and inflammation inhibition of wounds. Then, the curve values were maintained at a sustained release level, which was beneficial to cell proliferation. In addition, the benlysta-loaded SA hydrogel also had a slow degradation property, sill keeping 95% weight within 72 h. In combination with the drug release result, the hydrogel has good stability in a certain period of time, as shown in Figure .

Figure 4

Swelling rate of the benlysta-loaded hydrogel (mean ± SD, n = 3).

Figure 5

Benlysta release from the SA hydrogel (mean ± SD, n = 3).

Figure 6

Degradation detection of the benlysta-loaded SA hydrogel (mean ± SD, n = 3).

Biocompatibility of Benlysta-Loaded Hydrogel

To investigate the anti-inflammation property of the benlysta-loaded SA hydrogel, the macrophage test was performed because macrophage is the most important participation of inflammation. Figure shows that there are fewer macrophages in the benlysta-loaded SA hydrogel group compared with there are in the SA hydrogel group and the control group, suggesting the better anti-inflammation ability of the drug-loaded hydrogel.

Figure 7

(A) Acridine orange and ethidium bromide (AO/EB) staining images and (B) CCK-8 detection of macrophages in the benlysta-loaded SA hydrogel group and controls (***p < 0.001 compared with other samples, mean ± SD, n = 3).

Fibroblasts are the main components of deep connective tissue of the skin.[31]Figure shows that both SA hydrogel and benlysta-loaded SA hydrogel improved the fibroblast (L929 cell line) proliferation on the third day, wherein the benlysta-loaded SA hydrogel possessed a higher number of L929 compared with the single SA hydrogel, which indicated that the drug benlysta had a strong role in improving the fibroblast growth. Epidermal cells are the important outermost barrier of the skin.[32]Figure shows that the SA hydrogel and the benlysta-loaded SA hydrogel also improve the epidermal cell (HaCat cell line) proliferation on the third day. It is notable that the benlysta-loaded SA hydrogel as early as the first day began to improve the Faster HaCat growth, suggesting a better function in promoting skin epidermal healing.

Figure 8

(A) AO/EB staining images and (B) CCK-8 detection of L929 cell line in the benlysta-loaded SA hydrogel group and controls (*p < 0.05 compared with other samples, &p < 0.05 compared with the control group, mean ± SD, n = 3).

Figure 9

(A) AO/EB staining images and (B) CCK-8 detection of HaCat cell line in the benlysta-loaded SA hydrogel group and controls (*p < 0.05 compared with other samples, &p < 0.05 compared with the control group, mean ± SD, n = 3).

Many healing defects of skin damage caused by major diseases are due to the lack of blood vessels in the damaged tissue, which cannot provide enough nutrition, related factors, cells, etc. Thus, the vascularization of the focus is very important for the repair of damaged skin. Endothelial cells are the key components of blood vessels and play an important role in the process of tissue engineering vascularization.[33] In the present work, endothelial cells were seeded on the surface of the benlysta-loaded SA hydrogel, the single SA hydrogel, and the control group to evaluate the vascularization ability of each group. The fluorescence images (Figure A) and counting results (Figure B) showed that both the benlysta-loaded SA hydrogel and the single SA hydrogel promote endothelial cells growth, wherein the benlysta-loaded SA hydrogel showed higher number of endothelial cells. In addition, the endothelial cells on the benlysta-loaded SA hydrogel had a higher spreading area compared to the cells on the single SA hydrogel and the control group. All these results indicated that the benlysta-loaded SA hydrogel had a better ability to improve tissue engineering vascularization.

Figure 10

(A) Phalloidin (cytoskeleton) and 4,6-diamino-2-phenyl indole (DAPI) (nucleus) staining images and (B) counting results of endothelial cells in the benlysta-loaded SA hydrogel group and controls (*p < 0.05 compared with other samples, &p < 0.05 compared with the control group, mean ± SD, n = 3).

Conclusions

In this work, we prepared a benlysta-loaded SA hydrogel for anti-inflammation and promoting the growth of skin cells. The results of gelation experiment indicated that the benlysta-loaded SA hydrogel was time-dependent, and the gelation time could be controlled by the SA ratio, which promised potential application both as skin dressing and injectable subcutaneous material. The systematic material characterization also showed that the benlysta-loaded SA hydrogel had good water content, swelling rate, drug release function, and stability. Cell experiments showed that the benlysta-loaded SA hydrogel could inhibit the growth of macrophages and promote the growth of fibroblasts and skin epidermal cells. We hope this benlysta-loaded SA hydrogel may provide new ideas for treating skin injury that may be caused by external organic matter and/or major diseases.

Experimental Section

Preparation and Evaluation of Benlysta-Loaded SA Hydrogels

The preparation procedure of the SA hydrogels was the same as described in detail in the previous work.[19,34] In that study, benlysta was introduced into a SA hydrogel solution with a concentration of 320 mg/L before adding Ca2+ to trigger gelation. The gelation-forming process and the physical map of the benlysta-loaded SA hydrogels before and after the gelation were photographed using a HUAWEI MATE 10PRO mobile phone camera.[21] Material properties such as gelation time, water content, swelling rate, and degradation rate were characterized by our previous methods.[19,34] To investigate the anti-inflammatory function and pro-skin cell growth function of the benlysta-loaded SA hydrogels, macrophages, fibroblasts (L929 cell line), and epidermal cells (HaCat cell line) were seeded on the benlysta-loaded SA hydrogel, the single SA hydrogel, and the control surface and cultured for 24 and 72 h.[35] Then, the cells were stained with acridine orange and ethidium bromide (AO/EB) double-staining kit (Solabio) and observed by laser confocal microscopy (Nikon C2 Plus, Tokyo, Japan).[36] The CCK-8 kit was used to investigate cell proliferation, and the detected OD value at 450 nm had a positive correlation with the cell number.[37] To preliminarily evaluate the angiogenic ability of the benlysta-loaded SA hydrogel, endothelial cells, as an important part of blood vessels, were inoculated on the surface of the benlysta-loaded SA hydrogel, the single SA hydrogel, and the control group with a density of 4 × 104 cells/mL and then cultured under standard conditions for 24 and 72 h. After the endothelial cells had been washed with normal saline and fixed with 4% paraformaldehyde, they were stained with phalloidin and 4,6-diamino-2-phenyl indole (DAPI), and 15 pictures were taken for counting and statistics.[38]

Statistical Analysis

All of the values are presented as mean ± standard deviation using Origin8 software. Statistical analysis was performed by t test.