Gel loaded with spirulina powder nanoparticles as well as preparation method and application of gel

Abstract

The invention discloses a gel loaded with spirulina powder nanoparticles as well as a preparation method and application of the gel, edible spirulina powder is crushed, nanoparticles are extracted by using an ultracentrifugation method, and the nanoparticles are encapsulated in gel containing carboxymethyl chitosan, polyvinyl alcohol, polyvinylpyrrolidone and glycerol to prepare the gel loaded with the spirulina powder nanoparticles. The gel has good injectability and adhesion, and can effectively promote healing of oral ulcer. The gel has good biocompatibility, is simple and convenient to prepare, low in cost and easy for large-scale production, and has a wide application prospect in the field of oral ulcers.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a gel carrying spirulina powder nanoparticles, and a preparation method and application thereof. Background Art

[0002] Oral ulcers are a common oral mucosal disease, typically triggered by local trauma, nutritional deficiencies, immune abnormalities, or stress. They are characterized by pain, difficulty eating, and speech impairment. Existing treatments primarily include topical medications and systemic therapies (such as vitamin supplements), which can alleviate symptoms but are limited in effectiveness (Altenburg et al., 2014; Scully & Porter, 2008).

[0003] Spirulina, a spiral-shaped filamentous blue algae, is rich in protein, vitamins, minerals, antioxidants, and essential fatty acids, making it a valuable resource in both the food and pharmaceutical industries (Lafarga, Fernández-Sevilla, González-López, & Acién-Fernández, 2020; Maddiboyina et al., 2023). Studies have shown that spirulina is effective in treating various conditions, including metabolic syndrome, cardiovascular disease, and even cancer (Finamore, Palmery, Bensehaila, & Peluso, 2017; Krishnan et al., 2024; Prete et al., 2024; Wu et al., 2016). Furthermore, spirulina supplementation has been shown to improve lipid profiles and reduce oxidative stress in patients with hyperlipidemia (Torres-Duran, Ferreira-Hermosillo, & Juarez-Oropeza, 2007). However, the size of spirulina itself limits its absorption efficiency, which may hinder its effectiveness as a therapeutic agent. To overcome this limitation, it is necessary to develop spirulina nanoparticles. Spirulina nanoparticles have the potential to significantly enhance the bioavailability and cellular uptake of spirulina's active ingredients, ensuring more effective delivery to target tissues.

[0004] Chitosan oral gel has become an important option for the clinical treatment of oral ulcers. Its main components include chitosan as the active ingredient and excipients such as thickeners, stabilizers, and pH adjusters (Thanou, Verhoef, & Junginger, 2001). Chitosan is a polysaccharide derived from the deacetylation of chitin extracted from crustacean shells. It offers several benefits in the management of oral ulcers due to its biocompatibility, biodegradability, mucosal adhesion, and antimicrobial activity (Li et al., 2022; Wang et al., 2024). However, the therapeutic efficacy of chitosan oral gel may be limited by factors such as insufficient bioavailability and ineffective regulation of inflammation. Therefore, the addition of Spirulina nanoparticles may significantly enhance its effectiveness by overcoming these limitations and introducing additional pharmacological benefits. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide a gel containing spirulina powder nanoparticles and a preparation method and application thereof.

[0006] The object of the present invention is achieved through the following technical solution: a method for preparing a gel containing spirulina powder nanoparticles, comprising the following steps:

[0007] (1) Preparing a spirulina powder nanoparticle suspension: taking edible spirulina powder, dissolving it in a sterile liquid to obtain a spirulina powder solution with a concentration of 0.01 to 0.05 w / v; then adding the spirulina powder solution to a wall-breaking machine to break it up, centrifuging it at a speed of 6000 rpm for 20 minutes to remove the precipitate, collecting the supernatant, filtering it with a filter, and obtaining spirulina powder nanoparticles by a material separation method, which were resuspended in a sterile liquid to obtain a spirulina nanoparticle suspension;

[0008] (2) Preparation of a gel loaded with spirulina powder nanoparticles: first dissolve polyvinyl alcohol in sterile water at 80-90°C, then cool to room temperature, then add polyvinyl pyrrolidone, carboxymethyl chitosan, spirulina nanoparticle suspension, and glycerol in sequence, stir and mix thoroughly, centrifuge at 2000 rpm for 10 min to remove bubbles, and obtain a gel loaded with spirulina powder nanoparticles; the final concentration of the gel loaded with spirulina powder nanoparticles is as follows: polyvinyl alcohol 1.25% w / v, polyvinyl pyrrolidone 1.25% w / v, carboxymethyl chitosan 2.5% w / v, glycerol 2.0% v / v, and spirulina powder nanoparticles 20-200 μg / mL.

[0009] Furthermore, the sterile liquid is phosphate buffer, physiological saline or ultrapure water.

[0010] Furthermore, the substance separation method is ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity capture or filtration.

[0011] Furthermore, the centrifugal speed of the ultracentrifugation method is 100,000 g, and the centrifugation time is 120 min.

[0012] The invention also provides a gel carrying spirulina powder nanoparticles.

[0013] The present invention also provides an application of a gel carrying spirulina powder nanoparticles. The gel carrying spirulina powder nanoparticles is used for preparing a medicine for promoting the healing of oral ulcers.

[0014] Furthermore, the oral ulcer includes at least one of mild, severe, and herpes types.

[0015] Furthermore, the oral ulcer is located on the gums, hard palate, soft palate, buccal mucosa, inner lip, floor of mouth or tongue.

[0016] The beneficial effects of the present invention are:

[0017] 1) The present invention prepares a gel loaded with spirulina powder nanoparticles for the first time. The gel is injectable, viscous, and ductile, slowly releases the encapsulated drug, effectively promotes the healing of oral ulcers, and has good biocompatibility.

[0018] 2) The present invention utilizes a mixing method to prepare the gel and loads the nanoparticles derived from edible spirulina powder, which is simple to prepare, low in cost, and easy to mass-produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of spirulina powder and spirulina powder solution, wherein: Figure 1 (a) is a schematic diagram of Spirulina powder. Figure 1 (b) is a schematic diagram of the Spirulina powder solution;

[0020] Figure 2 This is an optical microscope image of Spirulina powder, where: Figure 2 (a) is an optical microscope image of Spirulina powder under the bright field channel. Figure 2 (b) is an optical microscope image of Spirulina powder under the chlorophyll fluorescence channel;

[0021] Figure 3 This is an optical microscope image of a spirulina powder solution, where: Figure 3 (a) is an optical microscope image of spirulina powder solution under the bright field channel. Figure 3 (b) is an optical microscope image of the spirulina powder solution under the chlorophyll fluorescence channel;

[0022] Figure 4 This is a transmission electron microscopy image of Spirulina powder nanoparticles;

[0023] Figure 5is the particle size distribution diagram of Spirulina powder nanoparticles;

[0024] Figure 6 This is the UV absorption spectrum of Spirulina powder nanoparticles;

[0025] Figure 7 Schematic diagram of the gel loaded with Spirulina powder nanoparticles;

[0026] Figure 8 This is a scanning electron micrograph of the gel loaded with Spirulina powder nanoparticles;

[0027] Figure 9 This is an experimental diagram of the injectability of the gel loaded with Spirulina powder nanoparticles;

[0028] Figure 10 Experimental diagram of the viscosity and ductility of the gel loaded with spirulina powder nanoparticles;

[0029] Figure 11 This is a release curve of the spirulina powder nanoparticles loaded gel slowly releasing spirulina powder nanoparticles in phosphate buffer at pH = 7.0;

[0030] Figure 12 This is a local image of a mild gingival ulcer in a rat treated with a gel containing spirulina powder nanoparticles;

[0031] Figure 13 This is a statistical diagram of the ulcer range of mild gingival ulcers in rats after being treated with a gel loaded with spirulina powder nanoparticles;

[0032] Figure 14 This is an H&E staining image of the local tissue section of the ulcer in a rat with mild gingival ulcer on the 8th day after being treated with the gel loaded with Spirulina powder nanoparticles;

[0033] Figure 15 This is a Masson staining image of the local tissue section of a mild gingival ulcer in rats after being treated with a gel loaded with Spirulina powder nanoparticles on the 8th day;

[0034] Figure 16 This is a local image of a severe ulcer on the mouse palate after being treated with a gel loaded with spirulina powder nanoparticles;

[0035] Figure 17 This is a statistical diagram of the ulcer range of severe ulcers on the upper palate of mice after being treated with a gel loaded with spirulina powder nanoparticles;

[0036] Figure 18 This is an H&E staining image of a local tissue section of a severe ulcer on the upper palate of a mouse treated with a gel containing spirulina powder nanoparticles on the 14th day;

[0037] Figure 19This is a Masson staining image of the local tissue section of a severe ulcer on the upper palate of a mouse treated with a gel containing spirulina powder nanoparticles on the 14th day;

[0038] Figure 20 H&E staining of sections of the heart, liver, spleen, lung, and kidney of a rat with mild gingival ulcer on day 8 after treatment with a gel loaded with Spirulina powder nanoparticles;

[0039] Figure 21 These are H&E-stained images of sections of the heart, liver, spleen, lung, and kidney of mice with severe palate ulcers 14 days after treatment with a gel loaded with Spirulina powder nanoparticles. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to illustrate the present invention, rather than to represent all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0041] The present invention provides a method for preparing a gel containing spirulina powder nanoparticles, comprising the following steps:

[0042] (1) Preparation of spirulina powder nanoparticle suspension: Edible spirulina powder is taken and dissolved in a sterile liquid to obtain a spirulina powder solution with a concentration of 0.01 to 0.05 w / v; the spirulina powder solution is then added to a wall breaking machine for crushing, centrifuged at a speed of 6000 rpm for 20 min to remove the precipitate, the supernatant is collected, filtered with a filter, and spirulina powder nanoparticles are obtained by a material separation method, which is then resuspended in a sterile liquid to obtain a spirulina nanoparticle suspension.

[0043] (2) Preparation of a gel loaded with spirulina powder nanoparticles: first dissolve polyvinyl alcohol in sterile water at 80-90°C, then cool to room temperature, then add polyvinyl pyrrolidone, carboxymethyl chitosan, spirulina nanoparticle suspension, and glycerol in sequence, stir and mix thoroughly, centrifuge at 2000 rpm for 10 min to remove bubbles, and obtain a gel loaded with spirulina powder nanoparticles; the final concentration of the gel loaded with spirulina powder nanoparticles is as follows: polyvinyl alcohol 1.25% w / v, polyvinyl pyrrolidone 1.25% w / v, carboxymethyl chitosan 2.5% w / v, glycerol 2.0% v / v, and spirulina powder nanoparticles 20-200 μg / mL.

[0044] The sterile liquid is phosphate buffer, physiological saline or ultrapure water.

[0045] The substance separation method is ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity capture or filtration.

[0046] The centrifugal speed of the ultracentrifugation method is 100000 g, and the centrifugation time is 120 min.

[0047] The spirulina powder nanoparticles prepared by the present invention have a size of nanometer level and a characteristic absorption spectrum. The diameter of the spirulina powder nanoparticles is 50 to 400 nm, and the absorption spectrum has a characteristic absorption peak at 627 nm.

[0048] The gel loaded with spirulina powder nanoparticles prepared by the present invention has a multi-pore structure with a pore size between 20 and 100 μm. The gel loaded with spirulina powder nanoparticles has good injectability, viscosity, and ductility, and has the effect of slowly releasing spirulina powder nanoparticles.

[0049] The spirulina powder-loaded nanoparticles prepared by the present invention have the efficacy of promoting the healing of oral ulcers and have good biosafety. The oral ulcers include at least one of mild, severe, and herpetic types, and are located in the gums, hard palate, soft palate, buccal mucosa, inner lips, floor of mouth, or tongue.

[0050] Example 1: Preparation and characterization of Spirulina nanopowder

[0051] Edible spirulina powder was taken and sterile PBS was used to prepare a spirulina powder solution with a concentration of 0.02 w / v. The spirulina powder solution was then added to a wall-breaking machine for crushing, and centrifuged at 6000 rpm for 20 minutes to remove the precipitate. The supernatant was collected and filtered through a 0.45 μm sterile filter. After ultracentrifugation at 100,000 g for 120 minutes, the spirulina powder nanoparticle precipitate was obtained, and the precipitate was resuspended in sterile liquid to obtain a spirulina nanoparticle suspension.

[0052] Figure 1 Schematic diagram of spirulina powder and spirulina powder solution, wherein: Figure 1 (a) is a schematic diagram of Spirulina powder. Figure 1 (b) is a schematic diagram of the Spirulina powder solution; Figure 2 This is an optical microscope image of Spirulina powder, where: Figure 2 (a) is an optical microscope image of Spirulina powder under the bright field channel. Figure 2 (b) is an optical microscope image of Spirulina powder under the chlorophyll fluorescence channel; Figure 3 This is an optical microscope image of a spirulina powder solution, where: Figure 3 (a) is an optical microscope image of spirulina powder solution under the bright field channel. Figure 3 (b) is an optical microscope image of the spirulina powder solution under the chlorophyll fluorescence channel.

[0053] The morphology of spirulina powder nanoparticles was detected using transmission electron microscopy. The results showed that spirulina powder nanoparticles contained a phospholipid bilayer spherical structure (similar to exosomes). In addition, there were various irregular nanoparticles, which might be protein structures or fragments (such as Figure 4 The particle size analysis results show that the particle size distribution of spirulina powder nanoparticles is mainly concentrated between 50-400nm (as shown). Figure 5 The UV absorption spectrum of the spirulina powder nanoparticles was detected by UV-visible-near infrared spectrophotometer, which showed a characteristic absorption peak at 627 nm (as shown in FIG. Figure 6 shown).

[0054] Figure 4 This is a transmission electron microscopy image of Spirulina powder nanoparticles. Figure 5 This is the particle size distribution diagram of Spirulina powder nanoparticles. Figure 6 This is the ultraviolet absorption spectrum of Spirulina powder nanoparticles.

[0055] Example 2: Preparation and characterization of spirulina powder-loaded nanoparticle gel

[0056] First, polyvinyl alcohol was dissolved in sterile water at 80°C and then cooled to room temperature. Then, polyvinyl pyrrolidone, carboxymethyl chitosan, the spirulina nanoparticle suspension prepared in Example 1, and glycerol were added in sequence. The mixture was thoroughly stirred and centrifuged at 2000 rpm for 10 min to remove bubbles. The spirulina powder nanoparticle gel (such as Figure 7 The final concentration of the obtained spirulina powder nanoparticle gel was 1.25% w / v polyvinyl alcohol, 1.25% w / v polyvinyl pyrrolidone, 2.5% w / v carboxymethyl chitosan, 2.0% v / v glycerol, and 100 μg / mL spirulina powder nanoparticles. Figure 7 Schematic diagram of the gel loaded with Spirulina powder nanoparticles.

[0057] The pore size morphology of the spirulina powder nanoparticle gel was detected by scanning electron microscopy. The results showed that the spirulina powder nanoparticle gel was porous and the pore size of the spirulina powder nanoparticle gel was between 20 and 100 μm (e.g. Figure 8 The spirulina powder nanoparticle gel was then transferred into a syringe to test the injectability of the spirulina powder nanoparticle gel. The results showed that the hydrogel system could be injected into any shape and maintained (as shown in FIG. Figure 9 The spirulina powder nanoparticle gel was placed on the skin to test the viscosity and ductility of the spirulina powder nanoparticle gel under different motion states (as shown). Figure 10 As shown). Figure 10 As shown, the gel has good viscosity and ductility and is suitable for use on soft tissue wounds.

[0058] Figure 8This is a scanning electron micrograph of the gel loaded with Spirulina powder nanoparticles. Figure 9 Experimental diagram showing the injectability of gel loaded with Spirulina powder nanoparticles. Figure 10 Experimental diagram of the viscosity and ductility of the gel loaded with spirulina powder nanoparticles.

[0059] Example 3: In vitro drug release performance testing

[0060] The spirulina powder nanoparticle gel prepared in Example 2 was added to 2 mL of phosphate buffer (pH = 7.0) and shaken at 37°C (100 rpm / min). The supernatant was collected at 0, 0.5, 1, 2, 4, 6 or 8 h, and the protein content in the supernatant was detected using BCA reagent. The in vitro release curve of the spirulina powder nanoparticles was calculated and plotted based on the initial total amount ( Figure 11 ). Figure 11 The release curve of the gel loaded with spirulina powder nanoparticles slowly releasing spirulina powder nanoparticles in phosphate buffer at pH = 7.0. Figure 11 As shown, it shows that the spirulina powder-loaded nanoparticle gel can slowly release nanoparticles within 12 hours.

[0061] Example 4: Therapeutic effect of spirulina powder-loaded nanoparticle gel on mild oral ulcers in rats

[0062] After anesthesia, rats were burned on the lower gums with a burner to form a mild oral ulcer with a diameter of 0.4 cm. 100 μL of the spirulina powder-loaded nanoparticle gel prepared in Example 2 was injected into the ulcer surface using a syringe and lightly applied to adhere to the ulcer surface. This was used once a day. The control group was not treated with drugs. The ulcers were photographed on days 0, 1, 2, 3, 4, 5, 6, 7, and 8 of treatment to detect and evaluate the healing rate. On day 8, the animals were euthanized, and local ulcer tissue was removed for pathological sectioning. H&E and Masson staining were performed to evaluate the ulcer healing effect.

[0063] The experimental results showed that compared with the control group, the use of spirulina powder nanoparticle gel significantly accelerated the healing of mild oral ulcers in rats. On the 8th day, the wounds in the experimental group had completely healed, while the wounds in the control group had not yet healed (e.g. Figure 12 and Figure 13 The results of H&E and Masson staining showed that the ulcer surface of the experimental group was completely re-epithelialized, while the ulcer surface of the control group was not yet completely covered by epithelial cells (as shown in Figure 2). Figure 14 and Figure 15 shown).

[0064] Figure 12 This is a local picture of a mild gingival ulcer in a rat after being treated with a gel loaded with spirulina powder nanoparticles. Figure 13This is a statistical chart of the ulcer range of mild gingival ulcers in rats after being treated with gel loaded with Spirulina powder nanoparticles. Figure 14 This is the H&E staining image of the local tissue section of the ulcer in a rat with mild gingival ulcer on the 8th day after being treated with the gel loaded with Spirulina powder nanoparticles. Figure 15 This is a Masson staining image of the local tissue section of the ulcer in a rat with mild gingival ulcer on the 8th day after being treated with the gel loaded with Spirulina powder nanoparticles.

[0065] Example 5: Therapeutic effect of spirulina powder-loaded nanoparticle gel on severe oral ulcers on the upper palate of mice

[0066] After anesthesia, mice were burned on the upper palate with a cauterizer to form a severe oral ulcer with a diameter of 1.5 cm. 200 μL of the spirulina powder nanoparticle gel prepared in Example 2 was injected into the ulcer surface using a syringe and lightly applied to adhere to the ulcer surface. This was used once a day. The control group was not treated with drugs. The ulcers were photographed on days 0, 2, 4, 6, 8, 10, 12, and 14 of treatment to detect and evaluate the healing rate. On day 14, the animals were euthanized, and local ulcer tissue was removed for pathological sectioning. H&E and Masson staining were performed to evaluate the ulcer healing effect.

[0067] The experimental results showed that compared with the control group, the use of spirulina powder nanoparticle gel significantly accelerated the healing of severe oral ulcers in mice. On the 14th day, the wounds in the experimental group had completely healed, while the wounds in the control group had not yet healed (e.g. Figure 16 and Figure 17 H&E and Masson staining results showed that the ulcers in the experimental group were completely epithelialized and the underlying connective tissue was connected together, while the control group still had a full-thickness defect (as shown in Figure 2). Figure 18 and Figure 19 shown).

[0068] Figure 16 This is a local image of a severe ulcer on the mouse palate after being treated with a gel loaded with Spirulina powder nanoparticles. Figure 17 This is a statistical chart of the ulcer range of severe ulcers on the mouse palate after being treated with a gel loaded with Spirulina powder nanoparticles. Figure 18 This is an H&E staining image of the local tissue section of the severe ulcer on the mouse palate 14 days after being treated with the gel loaded with Spirulina powder nanoparticles. Figure 19 This is a Masson staining image of the local tissue section of the severe ulcer on the mouse palate 14 days after being treated with the gel loaded with Spirulina powder nanoparticles.

[0069] Example 6: Safety testing of topical application of spirulina powder-loaded nanoparticle gel

[0070] After treating oral ulcers in rats and mice for 8 and 14 days respectively, the spirulina powder-loaded nanoparticle gel prepared in Example 2 was used to prepare pathological sections of important organs (heart, liver, spleen, lung, and kidney) and perform H&E staining. The experimental results showed that the histological structures of the heart, liver, spleen, lung, and kidney of the experimental group of rats and mice were no significantly different from those of the control group, indicating that the spirulina powder-loaded nanoparticle gel has good biosafety (such as Figure 20 and Figure 21 shown).

[0071] Figure 20 These are H&E staining images of sections of the heart, liver, spleen, lung, and kidney of rats with mild gingival ulcers 8 days after treatment with a gel loaded with Spirulina powder nanoparticles. Figure 21 These are H&E-stained images of sections of the heart, liver, spleen, lung, and kidney of mice with severe palate ulcers 14 days after treatment with a gel loaded with Spirulina powder nanoparticles.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a gel containing spirulina powder nanoparticles, characterized in that: The following steps are involved: (1) Preparing a spirulina powder nanoparticle suspension: taking edible spirulina powder, dissolving it in a sterile liquid to obtain a spirulina powder solution with a concentration of 0.01 to 0.05 w / v; then adding the spirulina powder solution to a wall-breaking machine to break it up, centrifuging it at a speed of 6000 rpm for 20 minutes to remove the precipitate, collecting the supernatant, filtering it with a filter, and obtaining spirulina powder nanoparticles by a material separation method, which were resuspended in a sterile liquid to obtain a spirulina nanoparticle suspension; (2) Preparation of a gel loaded with spirulina powder nanoparticles: first dissolve polyvinyl alcohol in sterile water at 80-90°C, then cool to room temperature, then add polyvinyl pyrrolidone, carboxymethyl chitosan, spirulina nanoparticle suspension, and glycerol in sequence, stir and mix thoroughly, centrifuge at 2000 rpm for 10 min to remove bubbles, and obtain a gel loaded with spirulina powder nanoparticles; the final concentration of the gel loaded with spirulina powder nanoparticles is as follows: polyvinyl alcohol 1.25% w / v, polyvinyl pyrrolidone 1.25% w / v, carboxymethyl chitosan 2.5% w / v, glycerol 2.0% v / v, and spirulina powder nanoparticles 20-200 μg / mL.

2. The method for preparing a gel containing spirulina powder nanoparticles according to claim 1, wherein: The sterile liquid is phosphate buffer, physiological saline or ultrapure water.

3. The method for preparing a gel containing spirulina powder nanoparticles according to claim 1, wherein: The substance separation method is ultracentrifugation, size exclusion chromatography, polymer precipitation, immunoaffinity capture or filtration.

4. The method for preparing a gel containing spirulina powder nanoparticles according to claim 3, wherein: The centrifugal speed of the ultracentrifugation method is 100000 g, and the centrifugation time is 120 min.

5. The gel containing spirulina powder nanoparticles prepared according to the method according to any one of claims 1 to 4.

6. Use of the gel containing spirulina powder nanoparticles according to claim 5, characterized in that: The gel loaded with spirulina powder nanoparticles is used for preparing medicine for promoting the healing of oral ulcers.

7. The use according to claim 6, characterized in that The oral ulcer includes at least one of mild, severe and herpes types.

8. The use according to claim 6, characterized in that The oral ulcer is located on the gums, hard palate, soft palate, buccal mucosa, inner lip, floor of mouth or tongue.

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