Chlorella-loaded sodium alginate microspheres as well as preparation method and application thereof

Abstract

The invention discloses a chlorella-loaded sodium alginate microsphere as well as a preparation method and application thereof, chlorella (MA) and sodium alginate are used as raw materials, and the chlorella-loaded sodium alginate microsphere is prepared by a microfluidic electronic injection technology. The prepared chlorella-loaded sodium alginate microspheres are intact in shape and can stably release oxygen. In-vivo and in-vitro experiments prove that the chlorella-loaded sodium alginate microspheres have a good wound treatment effect and have a huge clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a sodium alginate microsphere loaded with Chlorella vulgaris, its preparation method, and its application. Background Technology

[0002] Melanoma is highly aggressive and has a high potential for metastasis, making it one of the most deadly types of skin cancer. Surgical excision is currently the most common treatment for melanoma. However, surgical excision may be incomplete, and residual asymptomatic infiltrating tumor tissue often becomes a potential source of in situ recurrence or distant metastasis. Postoperative management of melanoma not only requires suppressing tumor recurrence and metastasis but also promoting the growth of normal tissue. Due to the weakened immune function of patients with malignant tumors, the full-thickness skin defects and large open wounds caused by surgery are difficult to heal, posing a major challenge to postoperative management of melanoma. Summary of the Invention

[0003] In view of the above, the purpose of this invention is to provide a method for preparing sodium alginate microspheres loaded with Chlorella alginate and its application. This invention uses Chlorella alginate (MA) and sodium alginate as raw materials and prepares Chlorella alginate microspheres using microfluidic electrospray technology.

[0004] The specific technical solution adopted in this invention is as follows: The first aspect of this invention provides a method for preparing sodium alginate microspheres loaded with Chlorella vulgaris, comprising the following steps: (1) Preparation of microfluidic device: Take a cylindrical capillary tube and use a stretcher to process one end of the circular glass capillary tube into a pointed cone shape. Use sandpaper to polish the inner diameter. Cut the other end to a suitable length and polish it smooth with sandpaper. Then fix the capillary tube on the glass slide, cover the capillary round end with the bottom of the needle and seal it with two-liquid hardening glue. Then use two-liquid hardening glue to stick the capillary body to the glass slide. (2) Preparation of sodium alginate microspheres loaded with Chlorella: Prepare a mixed solution of sodium alginate, Chlorella (MA) and vascular endothelial growth factor (VEGF), inject the mixed solution into a microfluidic device, remove the air in the microfluidic device, turn on the high voltage power supply for electro-spraying, cut out stable droplets, spray the droplets into a collection dish containing calcium chloride solution, and solidify the droplets into dispersed microspheres. The microspheres are the sodium alginate microspheres loaded with Chlorella.

[0005] Further, in step (1), the inner diameter is sanded to 100-200 μm using sandpaper.

[0006] Furthermore, the concentrations of sodium alginate and VEGF in the mixed solution are 2% w / w and 1‰ w / v, respectively, and the volume concentration of Chlorella (MA) is 1×10⁻⁶. 6 / mL.

[0007] Furthermore, the method for injecting the mixed solution into the microfluidic device is as follows: the mixed solution is drawn into a syringe and fixed on an infusion pump. The syringe needle is connected to a Teflon PFA tube, which is connected to the needle. A high-voltage power supply is connected to the metal part of the needle. The infusion pump parameters are set to inject the solution in the syringe into the microfluidic device at a uniform speed. After the air in the microfluidic device is expelled, the high-voltage power supply is turned on for electro-spraying to shear out stable droplets. The droplets are sprayed into a collection dish containing calcium chloride with a concentration of 2% w / w, so that the droplets solidify into dispersed microspheres.

[0008] The second aspect of the present invention is to provide sodium alginate microspheres loaded with Chlorella vulgaris, which are prepared by the above-described preparation method.

[0009] A third aspect of the present invention provides the application of the above-mentioned sodium alginate microspheres loaded with Chlorella vulgaris for wound healing.

[0010] Furthermore, the wound is a wound caused by surgical removal of melanoma.

[0011] The beneficial effects of this invention are as follows: Sodium alginate is a highly biocompatible polysaccharide extracted from seaweed that does not damage normal tissues. Chlorella (MA) has also been proven to have strong photosynthetic oxygen-releasing properties without causing harm to the body. The inventors discovered that by mixing sodium alginate with MA and adding VEGF to form a mixed solution, and by screening the proportions, microfluidic technology was used to fabricate morphologically intact sodium alginate microspheres loaded with Chlorella, which can stably release oxygen. Furthermore, both in vitro and in vivo experiments have demonstrated that these Chlorella-loaded sodium alginate microspheres have good wound healing efficacy and great clinical application potential. Attached Figure Description

[0012] Figure 1 Displayed as an electrospray-assisted microfluidic device for generating microspheres: digital image of the microfluidic device (a), microscopic image of the device (b); scale bar 420µm; Figure 2 The images shown are representative optical micrographs of sodium alginate microspheres (a) and sodium alginate microspheres loaded with Chlorella vulgaris (b), with a scale bar of 200 μm. Figure 3 The images shown are SEM images of sodium alginate microspheres and their surface (a) and SEM images of sodium alginate microspheres loaded with Chlorella vulgaris and their surface (b). The scale bar is 50 μm in the left image of (a) and (b) and 10 μm in the right image of (a) and (b). Figure 4 The images show SEM images of MA (a) and MA on the surface of the microspheres (b), with scale bars of 2 μm in (a) and 5 μm in (b). Figure 5 The particle size distribution is shown as that of sodium alginate microspheres loaded with Chlorella vulgaris. Figure 6 The image shown is a light micrograph of Chlorella vulgaris sodium alginate microspheres cultured for 7 days, with a scale bar of 150 μm. Figure 7 The data shows a quantitative analysis of oxygen changes in the blank control group and the group carrying sodium alginate microspheres of Chlorella vulgaris over 7 days (a) and an MA concentration of 1 × 10⁻⁶. 4 - 1 × 10 8 Quantitative analysis of oxygen changes in sodium alginate microspheres loaded with Chlorella at a density of 1 / mL (b); Figure 8 The images show fluorescence images of each group of materials after co-incubation with HSF under normal oxygen and low oxygen conditions (1% O2), stained with the low oxygen probe [Ru(dpp)3]Cl2 (red) and DAPI (blue), with a scale bar of 200 μm. Figure 9 The images show representative fluorescent staining images (a) and CCK-8 quantitative analysis (b) of HSF and each group of materials co-incubated for 5 days under hypoxic conditions (1% O2), with a scale bar of 200 μm. Figure 10 The images (a) and quantitative analysis (b) show representative images of in vitro scratch tests conducted under hypoxic conditions (1% O2) with HSF and various material groups. Dashed lines represent the initial scratch edges. The scale bar is 200 μm. Figure 11 The images shown are representative (a) and quantitative analysis (b) of HUVECs co-incubated with various materials under hypoxic conditions (1% O2), with dashed lines representing the initial scratch edges. Scale bar is 200 μm. Figure 12 The data shows the wound healing process of each group over 12 days (a), with a scale bar of 5 mm, and the quantitative data of wound closure at different time points (b), with N = 5 for each group; Figure 13 The images shown are representative HE staining images of each group on day 12; the dashed boxes indicate magnified areas, the scale bar (top) represents 500 μm, (bottom) represents 300 μm, and N = 5 for each group; Figure 14 The images show representative Masson trichrome staining (top) and immunofluorescence staining (CD31 / α-SMA, middle; HIF-1α, bottom) images of each group on day 12. Dashed boxes indicate magnified areas. Scale bars are 300 μm (top) and 100 μm (middle and bottom). Detailed Implementation

[0013] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0014] Unless otherwise specified, the materials used in the following embodiments are all commonly used materials in the art and are commercially available.

[0015] Example 1 A method for preparing sodium alginate microspheres loaded with Chlorella vulgaris includes the following steps: Fabrication of microfluidic devices: The electrospray-assisted microfluidic device is fabricated from a cylindrical capillary and a glass slide. The cylindrical capillary has an inner diameter of 0.8 mm and an outer diameter of 1 mm. One end is drawn into a single-ended cone shape using a pre-set capillary puller, and the inner diameter is sanded to 100-200 μm under an optical microscope. The other end is cut to a suitable length and smoothed. Residual debris inside the capillary is then ultrasonically cleaned, and the capillary is dried. The prepared capillary is placed centered and parallel on the glass slide, with the conical end extending beyond the slide. The rounded end and the capillary body are sealed with a needle and a two-component mixed hardening adhesive. After air drying overnight, debris is ultrasonically cleaned, and the capillary is stored in a dry, dust-free environment until use. Figure 1 (As shown).

[0016] Preparation of sodium alginate microspheres loaded with Chlorella vulgaris: Sodium alginate microspheres loaded with Chlorella vulgaris were prepared using sodium alginate, MA, and VEGF. The specific procedure is as follows: Sodium alginate (2% w / w) was prepared, MA (1×10⁻⁶) was added, and the microspheres were prepared. 6 A mixed solution of sodium alginate (1‰ w / v) and VEGF (1‰ w / v) was prepared. The solution was drawn into a 2.5 mL syringe and fixed to an infusion pump. The syringe needle was connected to a Teflon PFA tube, which in turn was connected to the device needle. A high-voltage power supply was connected to the metal part of the device needle. The infusion pump parameters were set to inject the solution from the syringe into the microfluidic device at a uniform rate. After the air inside the device was expelled, the high-voltage power supply was turned on, and electro-spraying was performed at an appropriate voltage to shear stable droplets. These droplets were sprayed into a collection dish containing calcium chloride (2% w / w) to solidify them into dispersed sodium alginate microspheres loaded with Chlorella. The morphology and particle size of the microspheres were observed under an optical microscope, and the experimental parameters were adjusted until the microspheres were uniform in size and stable in particle size.

[0017] Sodium alginate microspheres (sodium alginate (2% w / w) solution) were prepared using the method described in this embodiment. The morphology and particle size of the microspheres were observed under an optical microscope, and the experimental parameters were adjusted until the microspheres were of uniform size and stable particle size.

[0018] like Figure 2The image shows the light microscopic morphology of sodium alginate microspheres and sodium alginate microspheres carrying Chlorella vulgaris. Both are spherical. SEM was used to further characterize the complex details of the microstructure on the surface of the microspheres, such as... Figure 3 As shown, compared to sodium alginate microspheres, spherical MAs can be observed on the surface of sodium alginate microspheres carrying Chlorella vulgaris. Enlarging the SEM image, as shown... Figure 4 As shown in the figure, MA is uniformly distributed on the surface of the sodium alginate microspheres carrying Chlorella vulgaris. Figure 5 As shown, the sodium alginate microspheres loaded with Chlorella are uniform in size, with a particle size of approximately 200 μm.

[0019] Example 2 Photosynthetic oxygen release performance of sodium alginate microspheres loaded with Chlorella vulgaris: The distance between the LED lamp (6000 lux) and the culture dish carrying Chlorella alginate microspheres was fixed at 10 cm. Observations were conducted every two days, and the growth of *M. alginate* within the microspheres was observed and recorded using an optical microscope. Simultaneously, a dissolved oxygen meter was used to monitor the dissolved oxygen content in the Chlorella alginate microsphere culture medium under different experimental conditions. To assess its photosensitivity, the Chlorella alginate microspheres were exposed to LED light for 60 min, followed immediately by 60 min in complete darkness; this entire conversion culture process was repeated five times.

[0020] like Figure 6 As shown, a significant increase in MA can be observed under a microscope. On day 7, the growth of MA completely covered the inside and outside of the entire sphere, indicating that the MA in the microspheres prepared by microfluidic technology was not inactivated and had a strong survival and proliferation ability.

[0021] Following the preparation method described in Example 1, different MA concentrations (MA concentrations in the mixed solutions were 1 × 10⁻⁶) were prepared. 4 1 × 10 5 1 × 10 6 1 × 10 7 1 × 10 8 Sodium alginate microspheres loaded with *Chlorella vulgaris* (number of microspheres per mL) were used, and the dissolved oxygen (DO) level in the microsphere culture medium was monitored in real time over 7 days using a dissolved oxygen meter. Figure 7 As shown in Figure a, the DO value gradually increased with the increase of MA concentration in the microspheres, reaching a steady state after 4 days. Further quantitative evaluation of the photosynthetic oxygen release capacity of the microspheres at different MA concentrations was conducted. Figure 7 As shown in b, when the MA concentration is 1 × 10 6 The DO value reaches its highest level when the number of microspheres is 1 / mL, indicating that too little MA in the microspheres may not be able to release enough oxygen, while too much MA may be detrimental to their own growth and metabolism.

[0022] Example 3 In vitro cell experiments with sodium alginate microspheres from Chlorella vulgaris Different groups were set up: Ctrl group: control group, no treatment; MA group: Chlorella alginate microspheres loaded with sodium alginate (prepared according to the method of Example 1, the difference being that the mixed solution did not contain VEGF); MA-VEGF (dark) group: Chlorella alginate microspheres loaded with sodium alginate and VEGF cultured in the dark; MA-VEGF (light) group: Chlorella alginate microspheres loaded with sodium alginate and VEGF cultured under light. Simultaneously, normoxic HSF group: cell culture group under normal oxygen content; hypoxic HSF group: incubated in a hypoxic cell culture incubator (1% O2).

[0023] Cellular hypoxia relief experiment: The effect of oxygen-quenchable red fluorescent hypoxia indicator {[Ru(dpp)3]Cl2} on relieving intracellular hypoxia in cells loaded with sodium alginate microspheres from Chlorella vulgaris was evaluated. The normoxic group HSF was placed in 24-well plates (1×10⁻⁶). 5 The HSF from the hypoxia group was placed in 24-well plates (1×10⁶ cells / well) and incubated continuously for 12 h in a normoxic incubator. 5 Incubate continuously for 12 hours in wells containing sodium alginate microspheres (1×10⁻⁶ cells / well). Then, place the microspheres containing sodium alginate from Chlorella vulgaris (1×10⁻⁶ cells / well) into the wells. 6 (number / mL) and MA-VEGF-loaded sodium alginate microspheres (1×10⁻⁶) 6 Transwell chambers (number of cells / mL) were slowly placed into 24-well plates of HSF from normoxic and hypoxic groups, respectively, and incubated continuously for 12 h under normoxic and hypoxic conditions. The MA-VEGF sodium alginate microsphere-loaded group (MA-VEGF (Light)) was continuously irradiated under a 6000 lux LED lamp for 12 h. After incubation for 24 h under the above conditions, the Transwell chambers containing microspheres were removed from the 24-well plates, and the cell culture medium in each group was replaced with fresh DMEM medium containing 8 μg / mL of hypoxic probe. After incubation for 4 h under normoxic and hypoxic conditions, the DMEM medium containing the hypoxic probe in each 24-well plate was discarded. Subsequently, the cells were washed with fresh DMEM medium and fixed in 4% paraformaldehyde. Excess liquid in each 24-well plate was aspirated, and 4',6-diamidinyl-2-phenylindole (DAPI, blue) staining agent was added. The staining of cells in each group was observed under an inverted microscope.

[0024] like Figure 8As shown, the hypoxic fluorescence (red) in HSF co-cultured with the MA-VEGF (light) group was significantly reduced. This is very close to the results observed under normoxic conditions. The hypoxic fluorescence in HSF co-cultured with the MA group and the MA-VEGF (dark) group was also slightly reduced. However, the hypoxic fluorescence in HSF co-cultured with the Ctrl group remained almost unchanged. These results indicate that MA has the ability to release oxygen to alleviate cellular hypoxia; under light conditions, the oxygen release capacity of MA is significantly enhanced, and it can even reverse the cellular hypoxic state.

[0025] Cell proliferation experiment with sodium alginate microspheres of Chlorella: HSF from the hypoxia group was placed in a 24-well plate (1×10⁻⁶ m² / well). 3 Cells were incubated overnight in wells containing sodium alginate loaded with Chlorella vulgaris. Under a microscope, once cell growth was stable, the cells were placed in wells containing 1×10⁻⁶ cells of Chlorella vulgaris. 6 (number / mL) and MA-VEGF-loaded sodium alginate microspheres (1×10⁻⁶) 6 Transwell chambers (number cells / mL) were slowly placed into 24-well plates and incubated under hypoxic conditions. The MA-VEGF (Light) group was continuously irradiated under a 6000 lux LED lamp for 12 hours daily. On days 1, 3, and 5 of HSF co-culture with the cells, equal volumes of CCK-8 reagent were added to each well of the 24-well plates for each group of cells, ensuring sufficient contact with the cells, and then incubated in a hypoxic incubator for 30 minutes. Subsequently, equal volumes of CCK-8 reagent from each group were transferred to 96-well plates, and absorbance was measured to analyze cell proliferation over 5 days. The CCK-8 reagent was discarded, and HSF activity was observed using calcein (AM) and propidium iodide (PI) double staining.

[0026] If Figure 9 As shown in the figure, HSF cells co-cultured with the MA-VEG (light) group for 5 days showed significant cell proliferation, and HSF cells co-cultured with the MA-VEGF (dark) group for 5 days also showed some degree of proliferation, while HSF cells co-cultured with the MA group for 5 days showed no significant change. This indicates that MA has a strong ability to promote cell proliferation under light conditions, and VEGF also promotes cell proliferation to some extent.

[0027] Cell scratch assay of Chlorella alginate microspheres: HSF in the hypoxia group was placed in a 24-well plate (1×10⁻⁶ m² / well). 3 Cells were incubated overnight in wells (1 × 10⁶ cells / well). Under a microscope, once the cells had stabilized and formed a monolayer covering the bottom of the plate, the monolayer was scratched with a sterile pipette tip and washed twice with sterile PBS to remove any non-adherent cells. Subsequently, cells containing sodium alginate microspheres (1 × 10⁶ cells / well) were added to the plate. 6(number / mL) and MA-VEGF-loaded sodium alginate microspheres (1×10⁻⁶) 6 Transwell chambers (cells / mL) were slowly placed into 24-well plates and incubated under hypoxic conditions. The MA-VEGF (Light) group was continuously irradiated under a 6000 lux LED lamp for 6 h. Cell images of the lower chambers of the 24-well plates were acquired at 0 h, 12 h, and 24 h, and the relative migration area was calculated as: relative migration area (%) = (1 − W / W0) × 100%, where W represents the wound area at a specific time point, and W0 represents the wound area immediately after scratching. All data were acquired and processed using ImageJ software.

[0028] The results are as follows Figure 10 As shown, compared with the Ctrl group, HSF cells co-cultured with the MA-VEGF (light) group for 24 h exhibited very significant cell migration, and HSF cells co-cultured with the MA-VEGF (dark) group for 5 days also showed significant migration, while HSF cells co-cultured with the MA group for 5 days showed no significant change. This indicates that MA has a strong cell migration-promoting ability under light conditions, and VEGF also has a cell migration-promoting effect.

[0029] Angiogenesis assay of Chlorella vulgaris sodium alginate microspheres: HUVECs were resuscitated and expanded to a sufficient quantity in T25 and T75 cell culture flasks. Simultaneously, Matrigel was thawed, aliquoted, and uniformly added to 24-well plates under low-temperature conditions (on ice) to evenly coat the bottom of the wells. All wells were incubated at 37°C for 30 min to allow the gel to solidify. HUVECs were then seeded into 24-well plates (3 × 10⁶ cells / well) after Matrigel deposition. 5 (each well) contains sodium alginate microspheres loaded with Chlorella vulgaris (1×10⁻⁶). 6 (number / mL) and MA-VEGF-loaded sodium alginate microspheres (1×10⁻⁶) 6 Transwell chambers (number per mL) were co-cultured under hypoxic conditions for approximately 4 h. Excess liquid in the wells was discarded, and the lumen formation of the human umbilical vein endothelium was observed using calcein (AM) and propidium iodide (PI) double staining. The data were then processed using ImageJ software.

[0030] The results are as follows Figure 11 As shown, compared with the Ctrl group, HUVECs co-cultured with the MA-VEGF (light) group exhibited more angiogenesis, and HUVECs co-cultured with both the MA and MA-VEGF (dark) groups for 5 days also showed partial angiogenesis. This indicates that MA has a strong ability to promote angiogenesis under light conditions, and VEGF also shows the ability to promote angiogenesis.

[0031] Example 4 In vivo wound healing experiment of Chlorella alginate microspheres Under anesthesia, all fur on the backs of rats was shaved to expose the skin. A circular, full-thickness skin wound with a diameter of 1.5 mm was prepared on the back of each rat using a surgical scalpel. All rats were randomly divided into a control group, an MA group, an MA-VEGF (dark) group, and an MA-VEGF (light) group. Preparation methods for each group were as described above, with 6 rats in each group. PBS solution was applied to the skin surface of rats in the Ctrl group, MA microspheres were applied to the skin surface of rats in the MA group, MA-VEGF microspheres were applied to the skin surface of rats in the MA-VEGF (dark) group and they were kept in darkness, while MA-VEGF (light) group rats were applied to the skin surface and continuously irradiated under a 6000 lux LED bulb for 6 hours every 2 days. All rats were provided with ample food and water and kept in a dry, well-ventilated, and clean environment to ensure consistent external conditions and minimize interference from other factors. No other treatments or interventions were performed on the wounds except for the applied materials. Images of the rat dorsal wounds were collected on days 0, 2, 6, 10, and 12 after wound treatment. The wound size was measured using ImageJ, and the data were compared and processed.

[0032] The results are as follows Figure 12 As shown in figure a, compared with the Ctrl group, MA group, and MA-VEGF (dark) group, the MA-VEGF (light) group showed better wound healing, with the smallest wound area on day 12, achieving the best healing effect. Figure 12 Quantitative analysis showed that the MA-VEGF (light) group had the fastest healing process after different treatments in mice.

[0033] All rats were sacrificed on day 12, and wound tissue was collected and fixed with 4% paraformaldehyde. All tissue slides were dehydrated with ethanol, cleared, embedded in paraffin, and sectioned.

[0034] Complete tissue sections were selected for H&E staining analysis, and the results are as follows: Figure 13 As shown, compared with the Ctrl group, MA group, and MA-VEGF (dark) group, the MA-VEGF (light) group showed a very obvious epidermal healing layer and hair growth around the wound, while the other groups only showed a partial epidermal healing layer and no hair growth.

[0035] Complete tissue sections were selected and Masson's trichrome staining was used to assess collagen growth. The results are as follows: Figure 14 As shown in figure a, collagen deposition was found to be significantly increased in the MA-VEGF (light) group compared to the Ctrl, MA, and MA-VEGF (dark) groups. Figure 14Quantitative analysis showed that, after different treatments in mice, the MA-VEGF (light) group exhibited the fastest collagen deposition. Subsequently, to assess angiogenesis, we performed CD31 / α-SMA immunofluorescence staining on rat wound tissue sections, as shown... Figure 14 As shown in Figure a, the expression levels of CD31 (red) and α-SMA (green) were low in the Ctrl and MA groups, indicating low levels of angiogenesis. The MA-VEGF (dark) group showed slightly elevated levels of CD31 and α-SMA, suggesting that VEGF may have a role in promoting angiogenesis. The MA-VEGF (light) group showed significantly enhanced staining of CD31 and α-SMA, suggesting that, in addition to VEGF promoting angiogenesis, the oxygen released by MA further improved the hypoxic growth environment of cells, promoting angiogenesis in the wound. Figure 14 Quantitative analysis showed that the MA-VEGF (light) group had the highest vascular distribution compared to other groups, indicating that MA and VEGF significantly and synergistically promoted angiogenesis under light irradiation. Finally, the improvement of the hypoxic environment of cells at the wound site was assessed by HIF-1α specific antibody immunofluorescence staining. Results are as follows... Figure 14 As shown in Figure a, the MA group and the MA-VEGF (dark) group slightly improved the cellular hypoxic environment, with a partial decrease in hypoxic fluorescence (red). The MA-VEGF (light) group, however, significantly improved the cellular hypoxic environment, with a marked decrease in hypoxic fluorescence. Figure 14 Quantitative analysis showed that the MA-VEGF (light) group had the lowest hypoxia index, further proving that MA released a large amount of oxygen under light irradiation, which significantly improved the cellular hypoxic environment of rat wounds.

[0036] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing sodium alginate microspheres loaded with Chlorella vulgaris, characterized in that, Includes the following steps: (1) Preparation of microfluidic device: Take a cylindrical capillary tube and use a stretcher to process one end of the circular glass capillary tube into a pointed cone shape. Use sandpaper to polish the inner diameter. Cut the other end to a suitable length and polish it smooth with sandpaper. Then place the capillary tube flat on the glass slide, cover the capillary round end with the bottom of the needle and seal it with two-liquid mixed hardening glue. Then use two-liquid mixed hardening glue to stick the capillary body to the glass slide. (2) Preparation of sodium alginate microspheres loaded with Chlorella: Prepare a mixed solution of sodium alginate, Chlorella (MA) and vascular endothelial growth factor (VEGF), inject the mixed solution into a microfluidic device, remove the air in the microfluidic device, turn on the high voltage power supply for electro-spraying, cut out stable droplets, spray the droplets into a collection dish containing calcium chloride solution, and solidify the droplets into dispersed microspheres. The microspheres are the sodium alginate microspheres loaded with Chlorella.

2. The preparation method according to claim 1, characterized in that, In step (1), the inner diameter is sanded to 100-200 μm using sandpaper.

3. The preparation method according to claim 1, characterized in that, The concentrations of sodium alginate and VEGF in the mixed solution were 2% w / w and 1‰ w / v, respectively, and the volume concentration of Chlorella vulgaris (MA) was 1×10⁻⁶. 6 / mL.

4. The preparation method according to claim 1, characterized in that, The method for injecting the mixed solution into the microfluidic device is as follows: the mixed solution is drawn into a syringe and fixed to an infusion pump. The syringe needle is connected to a Teflon PFA tube, which is connected to the needle. A high-voltage power supply is connected to the metal part of the needle. The infusion pump parameters are set to inject the solution in the syringe into the microfluidic device at a uniform speed. After the air in the microfluidic device is expelled, the high-voltage power supply is turned on for electro-spraying to shear out stable droplets. The droplets are sprayed into a collection dish containing calcium chloride with a concentration of 2% w / w, so that the droplets solidify into dispersed microspheres.

5. A type of sodium alginate microspheres loaded with Chlorella vulgaris, characterized in that, It is prepared by the preparation method according to any one of claims 1-4.

6. The application of the sodium alginate microspheres loaded with Chlorella vulgaris according to claim 5, characterized in that, Medications used for wound healing.

7. The application according to claim 6, characterized in that, The wound was caused by surgical removal of a melanoma.

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