Multi-functional intelligent drug-loaded microalgae for the repair of diabetic wounds: oxygen supply, cell proliferation promotion, and hypoglycemic effect

Abstract

Diabetic wound healing has long been plagued by a series of complex problems caused by the pathological environment of high glucose, such as ischemia, hypoxia, and inflammatory responses. In order to solve this dilemma, we developed a new gel preparation with both green natural characteristics and excellent biological activity, aiming to provide an efficient and safe solution for diabetic wound healing. The gel uses microalgae as the core carrier, and it also plays an important role as an oxygen supply source. Through covalent bonding, the functional component concanavalin A and PEG-modified gold nanoparticles (PEG-AuNPs) were efficiently loaded on the polymer, which ensured the stable existence and controlled release of the components. Subsequently, the composite system was incorporated into the pre-gel fluid of the photocrosslinked methacryloylated gelatin to obtain our designed gel composite. On the one hand, the microalgae present in the material can continuously produce oxygen driven by light, effectively improve the local hypoxic microenvironment of the wound, and provide sufficient oxygen for cell proliferation and tissue repair. On the other hand, concanavalin A can specifically bind to glucose, and then cooperate with AuNPs with glucose oxidase activity to exert an efficient local hypoglycemic effect, thereby alleviating the adverse effects of high glucose on healing from the root. Through systematic experimental verification, this study confirmed the application prospects of this biocomposite material with multiple pro-healing properties in the field of diabetic wound management.

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1. Introduction

Diabetes, a chronic metabolic disorder, whose prevalence is increasing year by year, is one of the major diseases leading to increasing morbidity and mortality worldwide, and the number of affected patients is expected to increase to 643 million by 2030.^1–3 In clinical diagnosis and treatment, diabetic wounds such as diabetic foot are a major problem for doctors and patients, and severe cases that are not well controlled often lead to amputation. Normally, wound healing is a coordinated and orderly physiological dynamic process, but in diabetic wounds, the main cause of such severe results is the blockage of various normal physiological repair processes caused by the disorder of the wound microenvironment dominated by high glucose.^4,5 Upon hyperglycemic stimulation, glucose metabolism via the polyol pathway significantly increases, and this abnormal glucose metabolism inhibits the expression of antioxidants while inducing the formation of advanced glycation end products and promoting oxidative stress, which further aggravates inflammation.⁶ At the same time, hyperglycemia can lead to the damage of vascular endothelial cells, resulting in the expansion of the endothelial cell gap and enhanced permeability, which provide conditions for the subsequent inflammatory response and thrombosis. Hyperglycemia similarly leads to glycation of hemoglobin, vascular stenosis, and changes in the erythrocyte membrane, all of which lead to inadequate oxygen supply to wound tissue and vasculopathy.⁷ Similarly, in vitro studies have shown that glucose inhibits fibroblast and keratinocyte migration, further inhibiting epithelial regeneration.⁸ In conclusion, controlling the high glucose state of the diabetic wound and providing continuous oxygen supply to the wound is an essential treatment measure.

Synechococcus sp., as a typical single-cell rod-shaped microalga, has shown unique application potential in biomedicine.^9–11 It has been demonstrated that it can continuously release oxygen through photosynthesis in the in vitro environment, and this property can directly affect the wound repair process. It can not only significantly improve the local oxygenation level of the wound and provide sufficient oxygen supply for tissue regeneration, but also effectively promote the proliferation and migration of fibroblasts, accelerate the synthesis of collagen, and stimulate the activation of vascular endothelial cells and angiogenesis, thus promoting the process of wound healing.¹² Notably, the cell surface of microalgae is naturally enriched with a variety of chemical groups that can be used as biological binding sites, and these surface structures are often “decorated” by a series of natural functional groups. For example, the amino group (–NH₂) derived from protein molecules and the carboxyl group (–COOH) derived from sugar components such as glycoproteins and glycolipids on the cell membrane have good chemical reactivity and provide rich targets for subsequent biomolecular modification.¹³ Therefore, with their biological activity of continuous oxygen supply and modified surface chemistry, microalgae have become a promising “multifunctional carrier”, which provides an ideal natural platform for the construction of drug compound delivery systems that integrate oxygen supply, targeted delivery and controlled release.¹⁴

Concanavalin A (Con A) is a plant lectin extracted from the genus Concanavalin, and its binding ability to sugars has clear specificity and a molecular mechanism.^15,16 It has a significant reversible affinity for unmodified pyranose rings with hydroxyl groups at positions C-3, C-4, and C-6 in the glucose molecule. This specificity comes from the conserved sugar binding pocket in the structure of Con A protein, which forms a stable match with specific hydroxyl groups on the glucose pyranan ring through non-covalent interactions such as hydrogen bonds and van der Waals forces. Once these hydroxyl groups are modified (such as methylation, acetylation, etc.), or the conformation of the pyranan ring changes, their binding ability will be greatly reduced or would even disappear. It is this highly efficient and reversible recognition property of natural glucose molecules that makes Con A popular in the field of smart materials. Con A is often used as the core recognition element to build intelligent systems such as glucose-responsive drug delivery systems and blood glucose sensing devices.^17,18 Gold nanoparticles (AuNPs), as a kind of nanomaterial with unique physicochemical properties and biocompatibility, have attracted extensive attention in recent years for their enzyme mimetic activity. AuNPs can mimic the activity of glucose oxidase (GOx) under appropriate reaction conditions and can specifically catalyze the oxidation of glucose to produce gluconic acid and hydrogen peroxide, which are identical to the GOx catalytic products. This “nanoenzyme” property not only overcomes the defects of natural enzymes that are easy to inactivate and difficult to store, but also optimizes the catalytic efficiency by adjusting the size, morphology, surface modification and other parameters of AuNPs.^19,20 Meanwhile, the coordination property of nanoparticles has promoted their application in the biomedical field,^21–23 which has opened up a new direction for the development of personalized medicine.

Inspired by the above studies, we developed a multifunctional hydrogel for diabetic wound repair. The hydrogel was officially named Alg-Con-Au@Gel hydrogel (ACA@G). Con A with –NH₂ groups and PEG-AuNPs were bound to the microalgal surface by amide condensation reaction to form a whole complex.²⁴ When the glucose concentration is too high, Con A will bind to glucose and be stored in the surrounding solution, at which point AuNPs play their role as glucose oxidase-like enzymes to reduce the concentration of glucose around the wound. At the same time, the microalgae can supply oxygen through light. Meanwhile, we introduced the ACA complex into methylacrylylated gelatin containing photoinitiators. The gel can be formed rapidly under 405 nm light source irradiation (Scheme 1). This innovative method not only significantly accelerates the gelation rate, but also enables the hydrogel to form a gel structure directly on the wound surface in situ, which also gives the gel a rapid hemostasis effect. A series of in vitro and in vivo studies have confirmed that this integrated method has a variety of advantages such as lowering blood glucose, providing oxygen, and promoting angiogenesis and hemostasis, so that the multifunctional ACA@G can effectively deal with the key challenges of diabetic wounds, proving its great potential in clinical application.

Scheme 1 ACA@G gel preparation diagram and the working principle of promoting diabetic wound healing. The composite is composed of microalgae loaded with concanavalin A and PEG-AuNPs and applied via a photosensitive gel to a diabetic mouse wound model in vivo. It has been proven to regulate blood glucose levels, promote angiogenesis and hemostasis.

2. Results and discussion

2.1. Synthesis and characterization of ACA@G

We assembled concanavalin A (Con A) with –NH₂ groups and aminated PEG-AuNPs with microalgae through amide condensation reaction (Scheme 1). To explore the successful loading of the two material components, scanning electron microscopy images were taken, andas shown in Fig. 1A, Con A and PEG-AuNPs were effectively loaded on the microalgae (Con A: yellow arrow; PEG-AuNPs: red arrow). Biological scanning electron microscopy showed similar effects (Fig. 1B). As a kind of unicellular organism with chlorophyll, microalgae themselves can emit red fluorescence.²⁵ We stained concanavalin lectins using fluorescein isothiocyanate (FITC) fluorescent dye (green fluorescence). The results are shown in Fig. 1C, where laser scanning confocal microscopy showed that a certain amount of Con A was loaded on the microalgal surface, again confirming the effective loading of our added material. As shown in Fig. S1A, Fourier transform infrared spectroscopy (FTIR) showed good agreement between the enhancement of the amide signal observed at 1780–1700 cm⁻¹ at ACA and Alg-Con, thus confirming the amination reaction.^26,27 The zeta potentials of algae, Alg-Con and ACA complex particles were 0.07 mV, −0.04 mV and −0.05 mV, respectively, which also confirmed the successful loading of Con A and PEG-AuNPs on the microalgae (Fig. S1B). Subsequently, the in vitro oxygen release and glucose lowering properties of the materials were determined. As shown in Fig. S2A, the microalgae and ACA composite microalgae were able to continuously generate oxygen under different light intensities, and the oxygen production increased with increasing light intensity. As shown in Fig. 1D, both the ACA and the control gel alone were able to continuously supply oxygen within 6 h of light exposure, relative to the PBS group. At the same time, ACA was able to continuously produce oxygen during the process of light and dark transformation (Fig. S2B), which confirmed the continuous oxygen-producing performance and biological activity of microalgae. Similarly, the glucose-lowering performance of ACA was measured using a glucose kit, and the results showed that ACA was able to continuously reduce the concentration of glucose in solution from 16.6 mM to 10 mM within 36 h. The above results demonstrate the loading of AuNPs and the similar effect of glucose oxidase (Fig. 1E and S3).

Fig. 1 Characterization of the ACA. (A) SEM images of microalgae and different components loaded with microalgae, scale = 500 nm. (B) Biological electron microscopy image of ACA, scale bar = 2 μm and 1 μm. (C) Confocal image of fluorescently labeled Alg-Con (red: microalgae; green: Con A, scale = 500 nm). (D) Oxygen release curve of ACA under light within 6 hours. (E) Determination of the hypoglycemic effect of ACA. Data are presented as mean ± standard deviation (n = 3).

Subsequently, we performed a correlation assay on ACA@G gels, as shown in Fig. 2A, which showed rapid formation of gel complexes after 15 s of 405 nm illumination as compared with the control simple gel. SEM images of the lyophilized gel showed that the gel had a regular 3D porous structure (Fig. 2B). Gel rheology is the study of the mechanical properties of gels under external stress, with a focus on their viscoelasticity, yield behavior, and flow characteristics to understand the structure–property relationship, which is an indispensable part of the research on the biological application of gels.²⁸ The rheological properties of the hydrogel were examined using a rheometer. First, the viscosity of the ACA@G gel decreased with increasing shear rate as determined by shear thinning tests at 25 °C (Fig. 2C). The sol–gel transition process was determined by a strain amplitude scan test. Fig. 2D shows that for the ACA@G gel composite, the intersection between the storage modulus (G′) and the loss modulus (G′′) occurs at about 1000%. Continuous cyclic strain scans of 1% and 1000% were used to assess the self-healing performance of ACA@G (Fig. 2E). Under three changes of high and low strain, the hydrogel can transform between fluid and gel forms, indicating that it has good self-healing behavior. Although the rheological mechanical properties became worse compared to those of the gel alone (Fig. S4A–C), after three tests of high and low strain, the hydrogel fully recovered its modulus within 60 seconds without significant loss, thanks to the large number of groups in the modified gelatin, which fully demonstrated its excellent self-healing characteristics.²⁹

Fig. 2 Characterization of ACA@G. (A) ACA@G Body fluid before gel formation and the gel material after 405 nm blue light irradiation. (B) SEM of the ACA@G gel; scale = 100 μm. (C) Viscosity/shear rate measurements of ACA@G at 25 °C over a shear rate range of 0.1 to 10 rad s⁻¹. (D) Strain amplitude scanning test of ACA@G in the range of 1%–1000% scanning strain. (E) G′and G′′ changes between cyclic shear strain 1 and 1000% to verify the self-healing ability of ACA@G.

2.2. In vitro cell experiments of ACA@G

Severe diabetic wounds are usually accompanied by a state of high glucose and hypoxia, which seriously hinders the regeneration of some cells in the wound. We used ACA@G as an addition to the cell culture medium to perform the hypoglycemic and oxygen supplementation effects to explore the pro-proliferative effect in vitro. In this regard, we selected two kinds of cells closely related to skin wound repair: human umbilical vein endothelial cells (HUVEC) and fibroblasts (L929) for in vitro culture study. In order to clarify the biosafety of ACA@G and determine the optimal concentration of ACA@G, we performed CCK-8 cytotoxicity assessment using HUVEC cells as a model. The experimental results are shown in Fig. 3A. When the concentration of ACA@G in the medium was in the range of 0 to 24%, the effect on cell viability showed a significant concentration dependence: when the concentration of ACA@G was below 12%, the cell viability remained above 90%, and showed a slightly increasing trend with the increase of concentration, suggesting that there was no obvious toxicity in this concentration range of ACA@G, and it may even have a positive effect on cell survival. However, when the concentration reached 24%, the cell viability decreased sharply and obvious cell death occurred, which may be related to local microenvironment disturbance or excessive metabolic burden of materials at high concentrations. Based on this result, we uniformly set the ACA@G concentration to 12% in all subsequent cell experiments to ensure that its biological function was explored within a safe range. Subsequently, we set the glucose content of the medium to 33 mM and performed the experiments in a hypoxic incubator containing light. In Fig. 3B and D, the results show that the number of L929 cells in the ACA@G group was significantly higher than that in the control group. The same results could be seen in the Transwell cell migration assay. The cell migration distance and the number of cells passing through the membrane in the CA@G treatment group were significantly higher than those in the control group (Fig. 3C and E). This confirmed that ACA@G had a good ability to promote cell proliferation and migration in vitro.

Fig. 3 In vitro cell experiments at ACA@G. (A) ACA@G CCK-8 assay of HUVEC cells from 0% to 24% of the cell medium to determine the cytotoxic effect of the material. (B and D) Calcein-AM dye was used to detect the survival of L929 cells after treatment with different groups for 12 h, and their quantitative plots. Scale bar = 500 μm. (C and E) Results of Transwell assay and quantitative plots of L929 cells after treatment with different groups. Scale bar = 500 μm. Data are presented as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001.

2.3. Biocompatibility performance of ACA@G

Biocompatibility is the core prerequisite for the safe application of hydrogel materials in vivo, and related experiments are the key link to systematically evaluate their biocompatibility.³⁰ The cytocompatibility of the ACA@G gel was confirmed by the CCK-8 assay shown in Fig. 3A. In addition, considering that the gel needs to be in direct contact with blood in the region of wound repair, its blood compatibility is directly related to the safety of application. Therefore, ensuring its blood compatibility is essential for the safety of the application of the gel. After we mixed the hydrogel extract with blood cells, no obvious hemolytic reaction was observed (Fig. 4A). The hemolysis rate of hydrogels at all tested concentrations was below 1%, well below the critical threshold. We also determined the blood routine, liver function and renal function of the mice after the application of the ACA@G gel. As shown in Fig. 4B–F, there was no significant difference between the ACA@G gel group and the control group in all the corrected indicators of blood routine. Similar results were observed with measurements of liver and kidney function (Fig. S5A–F). Subsequently, H&E staining of the major organs (heart, liver, spleen, lungs, and kidneys) of mice was performed 48 h after the application of different groups of gel materials, and the results showed no obvious damage under the microscope (Fig. 4G). These results confirmed the good biocompatibility of ACA@G. For the ACA@G gel, no immune response was induced in the mice, probably because it was only used for wound indication, and the characteristics of microalgae themselves make it more like a “beneficial symbiotic partner” rather than an “enemy” to be eliminated.

Fig. 4 Biocompatibility experiments of ACA@G gels. (A) Hemolysis was determined by adding different concentrations of ACA@G (0–100 μL mL⁻¹) gels to PBS containing blood cells. (B–F) After the gel was applied to the wounds of mice, the differences of common blood routine values (RBC, WBC, PLT, HGB, and neutrophils) between ACA@G and the control group were compared. (G) H&E staining images of major mouse organs (heart, liver, spleen, lungs, and kidneys) after application of the ACA@G gel to mouse wounds. Scale bar = 200 μm. Data are presented as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, and ***P < 0.001.

2.4. The effect of the ACA@G gel on promoting wound repair in vivo

We performed in vivo experiments in mice to investigate the repair effect of the ACA@G gel. First, streptozotocin (STZ) was used to induce diabetes in male mice. When the blood glucose level exceeds 11.1 mM, it is regarded as a diabetes symptom.³¹ Subsequently, as shown in Fig. 5A, a mouse wound model with a diameter of 5 mm was made, and the mice were randomly divided into four groups to receive different treatments: the normal saline group (Control), simple gel group (Gel), microalgae gel group (Alg@Gel), and ACA@G (Alg-Con-Au@Gel) gel group. After the pre-gel droplets were added to the wounds of mice, the 405 nm light source was used to light cure the gel, so that the gel was attached to the wound to achieve the therapeutic effect. The dressing was changed every three days, and the gel was irradiated with light. The wounds were photographed and recorded. A significant reduction in wound size was observed in the Alg@Gel treatment group over a period of 12 days. The best healing was achieved on day 12, with a wound healing rate of 95%, whereas significant wounds were still present in the other groups (Fig. 5B and C).

Fig. 5 In vivo wound-healing performance assays for ACA@G. (A) Schematic diagram of a diabetic mouse wound model and experimental procedures. (B) Representative images of the wound healing process of different gel-treated mice on days 0, 3, 6, 9 and 12 and schematic diagram of the wound healing boundary, scale bar = 2 mm. (C) Corresponding quantitative analysis of wound closure, lasting for 12 days. (D) H&E stained sections of different treatments of skin tissue on days 6 and 12, scale bars = 1000 μm and 250 μm. (E) Masson stained sections of skin tissue from different treatments at days 6 and 12, scale bars = 1000 μm and 250 μm. Data are presented as mean ± standard deviation (n = 3); *P < 0.05, **P < 0.01, and ***P < 0.001.

In order to further explore the molecular mechanism of the ACA@G gel in promoting chronic diabetic wound healing, we performed RNA sequencing analysis on wound tissue samples collected from the ACA@G gel group and control group on day 6 of wound model establishment. Principal component analysis showed differential gene distributions between the two groups (Fig. S6A). In order to further analyze the differences of genes between the two groups, we performed a boxplot analysis, which allows us to not only view the dispersion of the gene expression level distribution of individual samples, but also visually compare the overall gene expression levels of the two groups of samples (Fig. S6B). As shown in Fig. 6A and B, overall, 1707 differentially expressed genes (DEGs) were identified between the gel group and the control group, of which 1120 were up-regulated and 587 were down-regulated. Subsequently, hierarchical clustering analysis was performed on the screened differentially expressed genes, and genes with the same or similar expression patterns were clustered. The clustering results of differentially expressed genes are shown in Fig. 6C, which confirmed that there were obvious differences between the two groups. To further explore the biological significance of these differentially expressed genes, GO enrichment analysis and KEGG enrichment analysis were performed to describe the genes and gene products related to glucose, oxygen and blood vessels in organisms, as well as to analyze gene function and genomic information data. Gene ontology analysis revealed functional differences in skin regeneration processes such as cell adhesion, angiogenesis, and wound healing (Fig. 6D). Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that ACA@G gel treatment significantly affected key signaling pathways related to cell adhesion, insulin resistance, inflammatory response, glucose metabolism, and glycation products (Fig. 6E). To further explore genetic differences, gene set enrichment analysis was performed for selected associated genes (Fig. S6C–E). This analysis revealed significant differences in cell respiration, glucose metabolism and cell adhesion regeneration between the two groups (P < 0.05).

Fig. 6 RNA sequencing analysis of diabetic wounds after 6 days of treatment with the ACA@G gel. (A) Comparison of gene expression differences between the experimental group treated with ACA@G and the control group. (B) Volcano plot analysis of gene expression differences between the experimental and control groups treated with ACA@G. (C) Heat map analysis of transcriptional expression profiles after ACA@G treatment. (D) GO enrichment analysis revealed differential gene expression in cell composition, molecular function and biological processes. (E) KEGG pathway enrichment analysis of differentially expressed genes.

Sequencing results showed that there were significant differences between the ACA@G gel-treated and control wounds at the genetic level. In this regard, the skin tissue was further detected and analyzed. H&E staining and Masson's trichrome staining (Fig. 5D and E) showed that at the initial stage of wound healing (day 6), there were no obvious signs of inflammatory response in the wounds of all hydrogel treatment groups. In sharp contrast, wound sites in controls showed numerous inflammatory cells with highly condensed chromatin, a feature typically associated with an active inflammatory response. As the healing process progressed, inflammatory cells decreased in all groups on day 13. Collagen density increased from day 6 to day 12 in all groups, with the ACA@G gel group exhibiting the highest collagen deposition (blue). In addition, the ACA@G gel group showed more blood vessels and hair follicles than the other groups at day 12. Collagen deposition, vascularization, and the formation of skin appendages such as hair follicles are essential for extracellular matrix remodeling and tissue regeneration during wound repair. According to the results, the ACA@G gel group showed the most complete and mature skin structure,³² the reason being that the ACA@G gel hydrogel can improve wound healing by reducing local blood glucose, promoting angiogenesis, and promoting cell migration.

In order to further confirm the ability of the ACA@G gel material to regulate the immune microenvironment of diabetic chronic wounds, we performed immunohistochemistry and fluorescent staining on the wound skin on the 6th and 12th days.

Vascular endothelial growth factor (VEGF) has been recognized as one of the most potent pro-angiogenic cytokines. Vascular growth is closely related to the expression of VEGF and its receptors, such as VEGFR1 and VEGFR2.³³ The pro-angiogenic effect of the material was investigated by measuring VEGF expression during the active phase of wound regeneration (day 6). As shown in Fig. S7A and B, a significant increase in the number of positive markers was observed from immunohistochemistry in the ACA@G treated tissues relative to the control group. The effective role of ACA@G in promoting angiogenesis was confirmed. Wound healing is a systematic, dynamic process involving epithelialization, angiogenesis, granulation tissue formation, and wound contraction, all of which are regulated by hypoxia-inducible factor 1-alpha (HIF-1α). Hyperglycemia will reduce the stability of HIF-1α and lead to the inhibition of HIF-1α target gene expression, which may be the cause of poor healing and ulcer complications in diabetic patients.^34,35 In response, we determined the expression of HIF-1α in different groups. On day 6, the ACA@G group had the highest expression of HIF-1α (Fig. S8A and B), which made the microenvironment attain a more stable state, confirming the role of ACA@G in promoting wound repair.

Advanced glycation (AGE) produced by hyperglycemia is an important diabetes-related endogenous risk related molecule, which can hinder diabetic wound repair.³⁶ Therefore, we investigated the hypoglycemic effect of ACA@G by measuring AGE. As shown in Fig. 7A and C, the positive markers observed by IHC in the ACA@G treated tissue were significantly attenuated at postoperative day 12. This indicates that ACA@G has a significant ability to remove inflammatory factors in the wound site and lower blood glucose, thereby reducing the harmful effects of oxidation and glycosylation. The formation of local new blood vessels is a key factor for diabetic wound healing. CD31, a member of the immunoglobulin superfamily, is mainly expressed on the surface of platelets, leukocytes, and endothelial cells. α-SMA is an actin isoform found mainly in vascular smooth muscle cells.³⁷ Therefore, immunofluorescence staining of CD31 and α-SMA was used to explore the promoting effect on wound neovascularization. As shown in Fig. 7B and D, the ACA@G group had the highest expression of CD31 and α-SMA by immunofluorescence staining, and had a larger lumen and more mature blood vessels.

Fig. 7 Histological analysis after application of the ACA@G gel to diabetic wounds. (A) AGE immunohistochemical staining of wound tissue on postoperative day 12, scale bar = 100 μm. (B) On the 12th day after the operation, CD31 and α-SMA immunofluorescence staining of the wound tissue, scale = 100 μm. (C) Quantitative map of AGE immunohistochemical staining of wound tissue on postoperative day 12. (D) Quantitative map of CD31 and α-SMA immunofluorescence staining in wound tissue on day 12. Data are presented as mean ± standard deviation (n = 3), *P < 0.05, **P < 0.01, and ***P < 0.001.

2.5. Hemostatic effect of the ACA@G gel

A photosensitive gel is an advanced hemostatic material that combines materials science and photochemistry. Its core mechanism can be summarized as follows: under the irradiation of a specific wavelength, the liquid or semi-solid gel precursor rapidly undergoes photopolymerization reaction, forming a solid physical barrier, so as to achieve rapid and firm hemostasis.^38,39 At the bleeding site, a liquid gel precursor, containing a photoinitiator, was overlaid on the wound. Being liquid, it penetrates and covers well the wounds that were irregular and sutured with difficulty. Subsequently, when illuminated with a specific light source, the photoinitiator absorbs light energy and initiates the polymerization reaction of monomer molecules, which transforms from a liquid to solid, elastic hydrogel network in tens of seconds.⁴⁰

To systematically evaluate the hemostatic efficacy of this hydrogel, a mouse liver hemorrhage model was constructed to simulate the common parenchymal organ hemorrhage scenario.⁴¹ In this study, partial hepatectomy was performed in mice. After continuous active bleeding occurred, the hydrogel precursor solution was evenly applied to the bleeding site and immediately irradiated with a 405 nm light source to trigger rapid gelation in situ. In this process, we used filter paper to record the amount of bleeding in areas such as the wound surface and the specific points where bleeding stopped, and comprehensively evaluated the hemostatic effect.

As shown in Fig. S9A, within 60 s after the start of the experiment, the wounds in the control group maintained obvious bleeding without significant signs of hemostasis. In sharp contrast, the bleeding of liver wounds treated with the hydrogel was effectively controlled in a short time, the bleeding scope was basically not expanded, and complete hemostasis and closure of wounds were achieved within 60 s. Blood loss was significantly less in the hydrogel-treated groups than in the control group throughout the hemostatic period (Fig. S9B and C). Similarly, as shown in Fig. S9D, total blood loss was significantly lower in the treatment group than in the control and control groups. The hemostasis time in the hydrogel group decreased significantly after the application of the photosensitive gel (Fig. S9E). These experimental results fully demonstrated the excellent hemostatic properties of the hydrogel in dealing with bleeding in parenchymal organs such as the liver.

3. Conclusions

In summary, we have developed a green microalga-driven light-responsive hydrogel designed to improve the delayed healing of diabetic chronic wounds. The microalgae were loaded with concanavalin lectins and PEGylated gold nanoparticles (PEG-AuNPs), making the hydrogel versatile. Both in vitro and in vivo experiments have confirmed that the ACA@G gel has excellent efficacy in alleviating hypoxia, lowering blood glucose, promoting angiogenesis and hemostasis, and accelerating diabetic wound healing, and has good biocompatibility. This study not only reveals an efficient drug loading delivery system, but also provides a new comprehensive treatment scheme for diabetic chronic wounds.

4. Experimental part

4.1. Materials

Microalgae (FACHB-805) were purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB). Concanavalin A, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. The amino-terminal PEGylated gold nanoparticles were purchased from Shanghai Dibai Biotechnology Co., Ltd. The live and dead cell staining kit, CCK-8 kit, and protein quantification kit (BCA) were purchased from Nanjing Bebo Biological Co., Ltd. Fluorescein 5-isothiocyanate (FITC) was purchased from KKL Med Inc., USA. Streptozotocin (STZ) and crystal violet stain were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Methacrylylated gelatin and photoinitiator LAP were purchased from EFL Inc. The glucose detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

4.2. Synthesis of the ACA@G gel

5 ml of the cultured microalgae were centrifuged and washed and dispersed in PBS. Then, according to a previous study,²⁴ 1 mL of 10 mg mL⁻¹ concanavalin A solution, 1.15 mg of EDC and 1.30 mg of NHS were added to the microalgal solution, and after stirring for 3 h, 1 mL of amino-modified PEG-AuNPs, 2.30 mg of EDC and 2.60 mg of NHS were added to the solution. After continuous stirring for 3 h, the modified microalgae were separated by differential centrifugation (3000 r, 5 min) and washed three times. The Alg-Con-Au complex was obtained.

Subsequently, 25 mg of LAP solution was added to 10 mL of PBS at 50 °C in the dark, and 500 mg of methacrylated gelatin was weighed and added to the above solution and stirred for 1 h at room temperature. The resulting solution was subsequently sterilized using a 0.22 μm sterile needle filter and stored away from light. The Alg-Con-Au complex described above was subsequently added to obtain ACA@G gel pre-solution.

4.3. SEM and biological electron microscopy

The relevant microalgae and their complexes were uniformly dispersed in PBS and subsequently drip-added onto a silicon wafer, and the solution was dried before being photographed under a transmission electron microscope. Before biological electron microscopy, the microalgal complexes were collected by centrifugation, placed in a 1.5 mL centrifuge tube, and fixed by adding 2.5% glutaraldehyde solution. The samples were then rinsed three times with 0.1 M phosphate buffer (pH 7.0) and fixed with 1% osmic acid solution for 1 h. The osmic acid waste solution was carefully removed, and the samples were rinsed three times with 0.1 M phosphate buffer (pH 7.0) for 15 min each time. The samples were dehydrated with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90% and 95%), and then treated twice with 100% ethanol. The samples were subjected to a graded solvent exchange, beginning with a 1 : 1 (v/v) ethanol/isoamyl acetate mixture for 30 min, followed by pure isoamyl acetate for 1 h or overnight. Subsequently, the samples were critically point dried, sputter-coated with a conductive layer, and examined by scanning electron microscopy (SEM).

4.4. Laser scanning confocal microscopy

FITC staining solution was prepared according to the FITC reagent instructions, and 2 μL of 5 mg mL⁻¹ FITC solution was added to 100 μl of 10 mg mL⁻¹ concanavalin A and incubated at 37 °C in the dark for 30 min. The microalgae and concanavalin A related recombination procedures were followed. After recombination, the microalgal solution was dropped onto the slide and flattened with a cover slip before being photographed under a confocal microscope.

4.5. Determination of oxygen release from microalgal complexes

To determine the oxygen-producing effect of microalgae in response to light, equal amounts of Alg-Con-Au mixture and microalgal solution were added into penicillin bottles and incubated under 1500 LUX illumination for 6 h. The oxygen content in the solution was measured at different time points using an oxygen electrode (JPBJ-608, Ritz, China). To determine the light-responsive oxygen release ability of microalgae, the microalgal solution was irradiated under light for 2 h, and then incubated in the dark for 2 h for three cycles. The oxygen content in the solution was measured using an oxygen electrode at different time points.

4.6. Determination of the hypoglycemic effect of microalgal complexes

A glucose solution at a concentration of 16.6 mM was configured, and the microalgal complexes described above were added to 5 mL glucose solution for incubation. At different time points (6, 12, 18, 24, and 36 h), the solution was taken for the determination of glucose. Glucose in the solution was determined using a glucose detection kit.

4.7. Rheological behavior determination of the ACA@G gel

The rheological behavior of the hydrogel was investigated using a rotational rheometer (MCR302e, Anton Paar, Austria). Prior to testing, each sample was equilibrated at 25 °C for 5 min by placing it in the middle of a 15 mm diameter parallel line. Viscosity measurements were performed at 25 °C in the range of 0.1 to 10 rad s⁻¹. Dynamic strain scan measurements were performed at a constant frequency of 10 rad s⁻¹ with the strain set in a range of 1–1000%. Then, the self-healing behavior of the hydrogel was evaluated by alternating step strain scan tests at a constant frequency (10 rad s⁻¹), in which the amplitude oscillatory strain was switched from low strain (1%) to high strain (1000%) 3 times with an interval of 60 s and an intermediate recovery of 10 s.

4.8. Live cell staining

L929 cells were seeded in 24-well plates, the medium was replaced with fresh medium (where the glucose content was set to 33 mM (ref. 37)) containing the various preparations described above (at a concentration of 12% Gel, Alg@Gel and Alg-Con-Au@G), and cultured under light. At predetermined time points (36 h), cells were washed with PBS and treated with calcein AM dye in the live/dead cell staining kit according to the manufacturer's protocol, and images were taken on an inverted microscope (Axio Observer 3, Zeiss, Germany) and quantified with ImageJ software.

4.9. Transwell cell migration assay

A 100 μL suspension of 5 × 10⁵ cells per mL of L929 cells was inoculated into the upper chamber, and the medium was replaced with fresh medium containing preparations from each group (where the glucose content was set to 33 mM). After 12 h of incubation in a light-exposed hypoxic incubator (5% oxygen concentration), cells were fixed with 4% formaldehyde for 30 min and washed three times with PBS. The cells were stained with 0.1% crystal violet for 15 min and washed three times with PBS. The unpenetrated cells in the upper chamber were stained with a cotton swab, and the migration of cells in the upper chamber was observed under an inverted microscope.

4.10. Hemolysis test

Eyeball blood was collected from Balb/C mice in anticoagulated vessels containing sodium citrate. After centrifugation at 3000 r for 15 min, 20 μL of blood cells were extracted and mixed with the following solution: various concentrations of ACA@G gels (25, 50, 75, 100 μL) were mixed with 1 mL of PBS, incubated at 37 °C for 4 h, and centrifuged at 3000 r for 15 min. Subsequently, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance at 542 nm was measured using a multifunctional enzyme analyzer to determine the hemolysis rate of the gels at different concentrations.

4.11. Biocompatibility of the ACA@G gel in animals

A 5 mm wound was made on the skin of healthy Balb/C mice. The gels of each group were dropped onto the back skin of mice, and a gel-like covering was formed on the wound after blue light irradiation. Heart, liver, spleen, lung, and kidney tissues and blood were collected from the mice after 24 hours, and the tissues were fixed in 4% formalin solution for further H&E section analysis. After the blood was centrifuged, serum was collected for blood biochemical tests, including routine blood test and liver and kidney function tests.

4.12. ACA@G gel for promoting diabetic wound healing

Male Balb/C mice were fed a high-fat, high-sugar diet and maintained in a specific pathogen-free environment. The mice were randomly divided into the control group, gel group, Alg@Gel group and Alg-Con-Au@Gel group. STZ (100 mg kg⁻¹) was mixed in sodium citrate buffer (intraperitoneal injection). Mice with a blood glucose level ≥11.1 mM 2 weeks after the first streptozotocin (STZ) injection under normal conditions were considered to have successfully induced diabetes. The animals were maintained in a diabetic state for wound healing experiments (3 times for diabetic mice). A full-thickness skin wound model with a diameter of 5 mm was made on the back of diabetic mice using a tissue biopsy device and scissors. The gel dressing was changed every 3 days and the gel was illuminated with light, and the wound area was photographed and recorded. Samples were taken on days 6 and 12 for further tissue characterization. The remaining wound area was measured using ImageJ software, and wound closure was calculated using the following equation.

Wound closure rate (%) = (A₀ − A_t)/A₀ × 100, where A₀ is the wound area on day 0 and A_t is the wound area A_t different time points.

At 6 and 12 days after surgery, the mice were sacrificed, and the tissue from the wound site was collected and fixed with 4% paraformaldehyde for histological analysis for hematoxylin and eosin (H&E) staining and Masson staining. Immunohistochemical analysis of VEGF and AGE and immunofluorescence staining of HIF-1α, CD31 and α-SMA were performed according to the standard protocol.^42,43

4.13. Hemostasis experiment

For the mouse hepatic hemorrhage model, mice were randomly divided into two groups (n = 3) and anesthetized with 3% sodium pentobarbital. The mice were rapidly immobilized before full exposure to the liver. A preweighed filter paper was placed below the liver prior to resection of a segment of liver tissue. After continuous active bleeding occurred, the hydrogel precursor solution was evenly applied to the bleeding site and immediately irradiated with a 405 nm light source to trigger the rapid gelation reaction in situ. Untreated mice were used as controls. The amount and time of bleeding were recorded through filter paper.

4.14. Transcriptome analysis

On day 6 after surgery, skin tissues were collected from the mice in the experimental group and the control group (n = 3 in each group) for subsequent RNA sequencing analysis. Total RNA was extracted using Trizol reagent. Subsequently, the quality of the resulting RNA was systematically assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA) and RNA-free agarose gel electrophoresis. After obtaining high quality total RNA, eukaryotic mRNA was enriched by Oligo(dT) beads. The enriched mRNA was interrupted into short fragments in fragmentation buffer and used as a template to construct a sequencing library using the NEBNext Ultra RNA Library Prep Kit for Illumina sequencing (New England Biolabs, Ipswich, MA). It was reverse transcribed into cDNA. Finally, all cDNA libraries were subjected to high-throughput sequencing on an Illumina Novaseq 6000 platform.

4.15. Statistical analysis

The results were expressed as mean ± standard deviation (mean ± SD). The Student's t-test was used for the comparison between two groups, and one-way analysis of variance (ANOVA) was used for the comparison among three or more groups. The survival curve was tested using the log-rank (Mantel–Cox) test. All statistical analyses were performed using GraphPad Prism software. Statistical significance is indicated as: *P < 0.05, **P < 0.01, and ***P < 0.001.

Author contributions

S. Y., N. J. and Y. X. have made equal contributions to this work. J. J., G. Z. and X. J. conceived and designed the project. S. Y. and N. J. carried out most of the experiments and analyzed the data. Y. X., L. H. and C. G. performed a part of the experiments. S. Y., N. J. and Y. X. drafted the manuscript. J. J. and G. Z. finished the manuscript.

Conflicts of interest

There are no conflicts to declare.

Ethical approval

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Anhui Medical University and were approved by the Animal Ethics Committee of Anhui Medical University. The animal study was approved by Anhui Medical University (Approval No: LLSC 20240664).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supplementary information (SI) is available. See DOI: https://doi.org/10.1039/d5bm01185j.

Acknowledgements

This research was supported by the Research Fund of Anhui Institute of Translational Medicine (No. 2022zhyx-C55) and Anhui Provincial Higher Education Quality Engineering Project (No. 2024aijy579). The schematic was created in https://BioRender.com.

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Footnote

† These authors have made equal contributions to this work.

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