Oil body loaded indocyanine green and imiquimod nanomicroparticles and applications thereof

Abstract

The application discloses oil body / indocyanine green / imiquimod microspheres, and a preparation method thereof, which comprises the following steps: 1) preparing an oil body suspension; 2) adding imiquimod, stirring, centrifuging, and reserving an oil body layer; 3) redissolving with dimethyl sulfoxide, adding an indocyanine green solution, stirring, and centrifuging to obtain the oil body layer, and the obtained oil body layer is the oil body / indocyanine green / imiquimod microspheres. Results show that the loading amount of R837 in safflower oil body is 1.51-18.87 mu g / mg; 808 nm near-infrared light excitation promotes the release of imiquimod, and the release efficiency reaches 60%; the oil body / indocyanine green / imiquimod microspheres can effectively inhibit tumors of tumor-bearing mice; the safflower oil body / indocyanine green / imiquimod microspheres have a large drug loading amount, can be absorbed through the skin or the intestinal tract, and have a good treatment effect on tumors.

Description

Technical Field

[0001] This invention belongs to the field of tumor drug technology, specifically relating to oil-loaded indocyanine green and imiquimod nanospheres and their applications. Background Technology

[0002] Melanoma, primarily affecting the skin, is a malignant tumor originating from malignant melanocytes. Its incidence rate is increasing by 3%-5% annually, making it one of the fastest-growing malignant tumors globally and the second leading cause of cancer death after leukemia. Statistics show that over 200,000 new cases are diagnosed worldwide each year, and melanoma accounts for approximately 80% of all skin cancer deaths. Furthermore, its incidence has been steadily increasing in recent years, making early diagnosis and treatment extremely important. In recent years, the number of malignant melanoma cases in my country has also increased significantly, with approximately 20,000 new cases annually. Currently, surgical resection is the primary treatment for cutaneous melanoma. For early-stage and locally advanced melanomas, surgery is easily performed and has a relatively good prognosis. However, for patients with advanced melanoma, surgical resection leads to a poor prognosis, low survival rate, and postoperative local complications, potentially causing severe functional impairment and cancer cell metastasis. Besides surgical resection, chemotherapy and radiotherapy are also common treatment strategies for tumors. However, long-term chemotherapy often involves severe side effects and the development of drug resistance; the clinical application of radiotherapy is severely limited by internal and environmental resistance. However, these treatments all have their drawbacks, such as patients' insensitivity to radiotherapy and chemotherapy, high rates of recurrence and metastasis after surgery, short duration of action of targeted drugs, easy development of drug resistance, and high treatment costs. Therefore, there is an urgent need to research various alternative therapies for melanoma.

[0003] Immunotherapy, as a novel treatment technology, has experienced rapid development over the past few decades. Imiquimod (R837), an immunomodulator approved by the U.S. Food and Drug Administration (FDA) that targets the Toll-like receptor 7, has been widely used to treat viral infections of the human anus and external genitalia, as well as skin cancers. However, R837 belongs to the nucleoside heterocyclic amine class of drugs, is insoluble in water and common organic solvents, and its formulation is difficult, thus limiting its widespread clinical application. Therefore, how to effectively deliver R837 as an immune adjuvant to the tumor site and release it effectively is a pressing problem to be solved in immunotherapy.

[0004] Oil bodies are spherical organelles with a diameter of 0.5–2.5 μm extracted from plant seeds. They consist of oil-binding proteins embedded in the surface, a phospholipid monolayer, and triglycerides encapsulated by the phospholipid monolayer. They possess advantages such as good physicochemical stability, low toxicity, and easy surface modification. Studies have found that oil bodies extracted from safflower seeds have good transdermal absorption capabilities, being completely absorbed by skin tissue within 30 minutes. Furthermore, due to the large number of hydrophobic triglyceride molecules within the oil body, they can bind hydrophobic drug molecules through hydrophobic interactions. Therefore, oil body materials can be used as transdermal drug delivery carriers for R837 in the treatment of cutaneous melanoma.

[0005] Furthermore, photothermal therapy is considered an effective treatment for tumors due to its non-invasive nature, simple procedure, high efficacy, negligible drug resistance, and few side effects. Indocyanine green (ICG), a typical cyanine dye approved by the FDA for clinical use, has been applied in the field of tumor photothermal therapy due to its good near-infrared light absorption properties. Summary of the Invention

[0006] The purpose of this invention is to provide safflower oil-supported indocyanine green and imiquimod nanospheres and their applications.

[0007] Oil-based / indocyanine green / imiquimod microspheres, prepared by the following method, including: 1) Preparation of oil suspension; 2) Add imiquimod, stir, centrifuge, and retain the oil layer; 3) Redissolve the microspheres in dimethyl sulfoxide, add them to the indocyanine green solution, stir, centrifuge, and obtain the oil layer. The oil layer obtained is the oil / indocyanine green / imiquimod microspheres. Step 1) Take 0.1-0.2g of the oil-based lyophilized powder and reconstitute it; 2) Add to 1 mL of imiquimod solution with a concentration of 1-20 mg / mL, stir at 0-4℃ in the dark for 2-4 h, centrifuge, and retain the oil layer; 3) Redissolve the oil layer with dimethyl sulfoxide, add it to 1 mL of indocyanine green solution with a concentration of 0.5-1.5 mg / mL, stir at 0-4℃ in the dark for 2-4 h, centrifuge, and the resulting oil layer is the oil / indocyanine green / imidaquimod microspheres. The concentration of the imiquimod solution mentioned in step 2) is 10-20 mg / mL; The concentration of the imiquimod solution mentioned in step 2) is 10 mg / mL; The concentration of the indocyanine green solution mentioned in step 3) is 1 mg / mL; The concentration of dimethyl sulfoxide mentioned in step 3) is 30%; The lyophilized oil powder mentioned in step 1) is obtained by thoroughly mixing safflower oil with mannitol and PBS buffer in a mass ratio of 2:1:7, followed by freezing and drying. The oil body mentioned is safflower oil body.

[0008] Application of oil-based / indocyanine green / imiquimod microspheres in the preparation of tumor-inhibiting drugs; The tumor in question is melanoma.

[0009] This invention provides safflower oil / indocyanine green / imiquimod microspheres. The preparation method includes: 1) taking 0.1-0.2g of lyophilized safflower oil powder and reconstituted it; 2) adding it to 1mL of imiquimod solution with a concentration of 1-20 mg / mL, stirring in the dark at 0-4℃ for 2-4h, centrifuging, and retaining the oil layer; 3) reconstituted the oil layer with dimethyl sulfoxide, adding it to 1mL of indocyanine green solution with a concentration of 0.5-1.5 mg / mL, stirring in the dark at 0-4℃ for 2-4h, centrifuging, and obtaining the oil layer as safflower oil / indocyanine green / imiquimod microspheres. The results show that the loading of R837 in safflower oil is 1.51-18.87 μg / mg; 808 nm near-infrared light excitation promotes the release of imiquimod, with a release efficiency of 60%. %; Safflower oil / indocyanine green / imiquimod microspheres can effectively inhibit tumors in tumor-bearing mice; the present invention has the following advantages: Safflower oil / indocyanine green / imiquimod microspheres have a large drug loading capacity, can be absorbed through the skin, and have a good therapeutic effect on tumors. Attached Figure Description

[0010] Figure 1 Microscopic images of safflower oil bodies and safflower oil body / indocyanine green / imiquimod microspheres; Figure 2 Absorption curves of safflower oil / indocyanine green / imiquimod microspheres before and after loading indocyanine green and imiquimod; Figure 3 Imiquimod release curves of safflower oil / indocyanine green / imiquimod microspheres; Figure 4 Photothermal images of indocyanine green and safflower oil / indocyanine green / imidaquimod nanospheres; Figure 5 Photothermal image under 808nm near-infrared light excitation; Figure 6 Curves showing the relative tumor volume changes in tumor-bearing mice after treatment with different materials. Detailed Implementation

[0011] Example 1: Preparation of freeze-dried safflower oil powder The specific steps for preparing lyophilized safflower oil powder are as follows: 1) Take 2g of safflower seeds after the shells have been broken, add 20 mL of PBS buffer (pH=7.4), grind thoroughly, and centrifuge at 12000 rpm for 5 min to obtain the oil layer; 2) Add PBS buffer to the oil layer again, mix well, and centrifuge at 12000 rpm for 5 min. Repeat the above operation until no precipitation is produced. The obtained oil layer is the safflower oil. 3) The obtained safflower oil body was thoroughly mixed with mannitol and PBS buffer at a mass ratio of 2:1:7, and then frozen at -80℃ for 12~24h. After that, it was transferred to a freeze dryer and freeze-dried for 36~48h to obtain safflower oil body freeze-dried powder.

[0012] Example 2 Preparation of Safflower Oil / Imiquimod Microspheres 0.15 g of lyophilized safflower oil powder was reconstituted with 0.2 mL of 30% acetic acid. After reconstitution, the solution was added to 1 mL of imiquimod solution (10 mg / mL), and the mixture was slowly stirred at 4 °C in the dark for 3 h. After centrifugation at 10,000 rpm for 5 min, the oil layer was retained to obtain safflower oil / imiquimod microspheres. The loading of R837 in the safflower oil was detected to be approximately 18.87 μg / mg.

[0013] Example 3: Preparation of Safflower Oil / Indocyanine Green / Imiquimod Microspheres Take 0.15 g of lyophilized safflower oil and reconstitute it with 30% acetic acid (0.2 mL). After reconstitution, add it to 1 mL of imiquimod solution (1 mg / mL), stir slowly in the dark at 4℃ for 3 h, centrifuge at 10000 rpm for 5 min, and retain the oil layer. Reconstitute the retained oil layer with 30% dimethyl sulfoxide and add it to 1 mL of indocyanine green solution (concentration 1 mg / mL). Stir slowly in the dark at 4℃ for 3 h, centrifuge at 8000 rpm for 3 min, and retain the oil layer to obtain safflower oil / indocyanine green / imiquimod microspheres. The loading of R837 in safflower oil was found to be approximately 1.51 μg / mg.

[0014] Example 4: Preparation of Safflower Oil / Indocyanine Green / Imiquimod Microspheres Take 0.15 g of lyophilized safflower oil and reconstitute it with 30% acetic acid (0.2 mL). After reconstitution, add it to 1 mL of imiquimod solution (5 mg / mL), stir slowly at 4℃ in the dark for 3 h, centrifuge at 10000 rpm for 5 min, and retain the oil layer. Reconstitute the retained oil layer with 30% dimethyl sulfoxide and add it to 1 mL of indocyanine green solution (concentration 1 mg / mL). Stir slowly at 4℃ in the dark for 3 h, centrifuge at 8000 rpm for 3 min, and retain the oil layer to obtain safflower oil / indocyanine green / imiquimod microspheres. The loading of R837 in safflower oil was found to be approximately 7.86 μg / mg.

[0015] Example 5: Preparation of Safflower Oil / Indocyanine Green / Imiquimod Microspheres 0.15 g of lyophilized safflower oil powder was reconstituted with 0.2 mL of 30% acetic acid. The reconstituted oil was then added to 1 mL of imiquimod solution (10 mg / mL), and the mixture was stirred slowly at 4°C in the dark for 3 h. After centrifugation at 10,000 rpm for 5 min, the oil layer was retained. The retained oil layer was then reconstituted with 30% dimethyl sulfoxide and added to 1 mL of indocyanine green solution (1 mg / mL). The mixture was stirred slowly at 4°C in the dark for 3 h, and after centrifugation at 8,000 rpm for 3 min, the oil layer was retained, yielding safflower oil / indocyanine green / imiquimod microspheres. The loading of R837 in the safflower oil was detected to be approximately 18.87 μg / mg.

[0016] Example 6: Preparation of Safflower Oil / Indocyanine Green / Imiquimod Microspheres 0.15 g of lyophilized safflower oil powder was reconstituted with 0.2 mL of 30% acetic acid. The reconstituted oil was then added to 1 mL of imiquimod solution (20 mg / mL), and the mixture was stirred slowly at 4°C in the dark for 3 h. After centrifugation at 10,000 rpm for 5 min, the oil layer was retained. The retained oil layer was then reconstituted with 30% dimethyl sulfoxide and added to 1 mL of indocyanine green solution (1 mg / mL). The mixture was stirred slowly at 4°C in the dark for 3 h, and after centrifugation at 8,000 rpm for 3 min, the oil layer was retained, yielding safflower oil / indocyanine green / imiquimod microspheres. The loading of R837 in the safflower oil was detected to be approximately 17.96 μg / mg.

[0017] Example 7: Tumor-killing effect of safflower oil / indocyanine green / imiquimod microspheres Safflower oil, the safflower oil / imidazolide microspheres prepared in Example 2, and the safflower oil / indocyanine green / imidazolide microspheres prepared in Example 5 were respectively placed in PBS buffer solution, and their images under a microscope were observed. The results are as follows: Figure 1 The materials were uniformly dispersed, indicating that the structure of safflower oil was not damaged before and after the synthesis.

[0018] Figure 2 The absorption curves of safflower oil / indocyanine green / imiquimod microspheres before and after loading indocyanine green and imiquimod indicate that safflower oil was successfully loaded with indocyanine green and imiquimod. To ensure successful release of imiquimod from safflower oil / indocyanine green / imiquimod, at a concentration of 0.4 W / cm 2 Irradiated with 808 nm near-infrared light for 5 min; the release effect of imiquimod was detected, and the release curve was plotted by measuring the concentration of imiquimod in the solution. Figure 3 ; Figure 3 The figure shows the imiquimod release curve of safflower oil / indocyanine green / imiquimod microspheres after 808 nm near-infrared light excitation. It can be seen from the figure that the longer the time, the higher the imiquimod release efficiency, indicating that 808 nm near-infrared light excitation promotes the release of imiquimod, with a release efficiency of 60%.

[0019] Figure 4 These are photothermal images of indocyanine green alone and safflower oil / indocyanine green / imiquimod nanospheres under 808 nm near-infrared light excitation. The results showed that safflower oil / indocyanine green / imiquimod rapidly reached 40 °C within 3 min, while indocyanine green alone could only rise to 32 °C. This indicates that loading indocyanine green onto safflower oil reduces heat loss and is beneficial for promoting photothermal therapy.

[0020] Figure 5 The images show photothermal images under 808 nm near-infrared light excitation after the tumor site of tumor-bearing mice absorbed safflower oil / indocyanine green / imiquimod nanospheres. The results showed that after irradiation at 0.4 W / cm2 for 5 min, the temperature at the tumor site increased to 42℃. This indicates that the safflower oil / indocyanine green / imiquimod nanospheres were successfully absorbed by the mice and successfully reached the melanoma site, generating a large amount of heat. This demonstrates that safflower oil is a good drug carrier for transdermal drug delivery.

[0021] Figure 6 The figures show the relative tumor volume changes in tumor-bearing mice after treatment with different materials. The results showed that the tumor was effectively inhibited in the safflower oil / indocyanine green / imiquimod photothermal treatment group. The safflower oil / indocyanine green / imiquimod non-photothermal treatment group also showed some tumor inhibition. This is because after entering the tumor site, safflower oil / indocyanine green / imiquimod released a small amount of imiquimod, which produced a certain immune-killing effect on the tumor, but the effect of single treatment was not significant. Combined photothermal treatment showed better tumor inhibition, effectively controlled tumor volume, and demonstrated a better tumor-killing effect, indicating that combined treatment can effectively improve anti-tumor ability.

Claims

1. Oil / Indocyanine Green / Imiquimod microspheres, prepared by the following method, including: 1) Prepare the oil suspension by taking 0.1-0.2 g of lyophilized oil powder and reconstitute it; 2) Add it to 1 mL of imiquimod solution with a concentration of 1-20 mg / mL, stir in the dark at 0-4℃ for 2-4 h, centrifuge, and retain the oil layer; 3) Reconstitute the oil layer with dimethyl sulfoxide, add it to 1 mL of indocyanine green solution with a concentration of 0.5-1.5 mg / mL, stir in the dark at 0-4℃ for 2-4 h, centrifuge, and the resulting oil layer is the oil / indocyanine green / imiquimod microspheres; The lyophilized oil powder mentioned in step 1) is obtained by thoroughly mixing safflower oil with mannitol and PBS buffer in a mass ratio of 2:1:7, followed by freezing and drying.

2. The oil / indocyanine green / imiquimod microspheres according to claim 1, characterized in that: The concentration of the imiquimod solution in step 2) is 10–20 mg / mL.

3. The oil / indocyanine green / imiquimod microspheres according to claim 2, characterized in that: The concentration of the imiquimod solution in step 2) is 10 mg / mL.

4. The oil / indocyanine green / imiquimod microspheres according to claim 3, characterized in that: The concentration of the indocyanine green solution in step 3) is 1 mg / mL.

5. The oil / indocyanine green / imiquimod microspheres according to claim 1, 2, 3 or 4, characterized in that: The concentration of dimethyl sulfoxide in step 3) is 30%.

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