Tilrolic acid nanostructured lipid carriers, preparation method and application thereof

Abstract

The application discloses a nano-structured lipid carrier and a preparation method and application thereof, and the preparation method comprises the following steps: heating, melting and mixing solid lipids, squalene and an emulsifier to form an oil phase, dissolving tirofiban in heated water to form a water phase; mixing the oil phase with the water phase, performing high-shear emulsification to obtain a coarse emulsion; performing ultrasonic treatment on the coarse emulsion to obtain a nano-emulsion; and shearing the nano-emulsion into ice water and cooling in an ice water bath. The average hydrated particle size of the nano-structured lipid carrier prepared by the application is 100-200 nm, the polydispersity coefficient is less than 0.25, and the zeta potential is-10 to-20 mV. The application has excellent drug loading and slow-release performance, the encapsulation rate of tirofiban reaches 89%, the slow release of the drug can be realized, the in-vivo action time of the drug is prolonged, and the application has remarkable antiviral activity.

Description

Technical Field

[0001] This invention pertains to the preparation of drug carriers, specifically a telotron nanostructure lipid carrier, its preparation method, and its application. Background Technology

[0002] Subunit vaccines are favored for disease control in livestock farming due to their high safety profile, but they generally suffer from weak immunogenicity and short duration of immunity, requiring highly effective adjuvants to enhance the immune response. Nanostructured lipid carriers (NLCs), as a new generation of antigen delivery systems, offer advantages such as high encapsulation efficiency, good stability, strong sustained release, and excellent biocompatibility. They can also protect antigens from enzymatic degradation, improve antigen solubility and bioavailability, and prolong drug release time, showing significant potential for transdermal drug delivery and vaccine administration.

[0003] Squalene, an immunomodulatory lipid, has been successfully used in the human adjuvant MF59. However, its application in veterinary vaccines is limited by its lack of strong immunostimulatory function, and squalene preparations alone cannot be stored for long periods. Tilololon, a synthetically produced small molecule compound, possesses broad-spectrum antiviral and immunomodulatory activities. Studies have shown that tilololon inhibits various viruses, including influenza virus. In a porcine alveolar macrophage (3D4 / 21) model, tilololon, within a safe concentration range, significantly inhibited the replication of swine influenza virus H3N2, reduced virus-induced cytopathic effects, and decreased the expression of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α. In a piglet infection model, gavage administration of tilololon reduced viral load in lung tissue and alleviated pulmonary inflammatory infiltration and pathological damage. Simultaneously, it can act as a Toll-like receptor 7 / 8 (TLR7 / 8) agonist, effectively inducing the production of type I interferon (IFN-α / β), bridging innate and adaptive immunity, and enhancing humoral and cellular immune responses. However, when tiloron is used directly as a small molecule drug, it has problems such as rapid clearance in the body, low bioavailability, and potential systemic toxicity, which limit the full realization of its adjuvant efficacy.

[0004] Chinese patent CN111494619A discloses a method for preparing a squalene-based cationic nanostructure lipid carrier immunoadjuvant. However, it only uses squalene as a lipid adjuvant component and does not simultaneously carry small molecule compounds with immune agonistic activity, resulting in drawbacks such as single adjuvant function, limited immune stimulation pathway, no antiviral activity, and poor biocompatibility. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing a nanostructured lipid carrier that is simple in preparation process, cost controllable, safe, and can be mass-produced and stored for a long time. Another purpose of this invention is to provide a nanostructured lipid carrier that has dual functions of antigen delivery and immune stimulation and can significantly enhance the humoral and cellular immune responses of vaccines. A further purpose of this invention is to provide an application of the nanostructured lipid carrier in the preparation of vaccine compositions.

[0006] Technical solution: The present invention provides a method for preparing a telotron nanostructured lipid carrier, comprising the following steps:

[0007] Step 1: Heat and melt the solid lipids, squalene, and emulsifier to form an oil phase; dissolve tilaron in pure water and heat in a water bath to form an aqueous phase.

[0008] Step 2: Mix the oil phase and the water phase, and perform high-shear emulsification to obtain a crude emulsion;

[0009] Step 3: The crude emulsion is subjected to ultrasonic treatment to obtain a nanoemulsion;

[0010] Step four: The nanoemulsion is transferred to ice water for shearing and cooled in an ice water bath to obtain nanostructured lipid carriers (tsNLCs).

[0011] Furthermore, in step one, the mass ratio of solid lipids, squalene, emulsifier, telotron, and water is 1:9:3:1:90~100.

[0012] Furthermore, in step one, the solid lipid is palmitic acid.

[0013] Furthermore, in step one, the emulsifiers are equal masses of Tween 80 and Span 85.

[0014] Furthermore, in step one, the heating temperature is 75~85℃.

[0015] Furthermore, in step two, the high-shear emulsification speed is 10,000~12,000 rpm, and the time is 3~10 minutes.

[0016] Furthermore, in step three, the ultrasonic treatment power is 200~400 W, and the time is 15~25 minutes.

[0017] The nanostructured lipid carrier prepared by the preparation method described in this invention has an average hydrated particle size of 100~200 nm, a polydispersity index of less than 0.25, and a zeta potential of -10~-20 mV.

[0018] This invention provides the application of a nanostructured lipid carrier in the preparation of vaccine compositions.

[0019] Furthermore, the vaccine composition includes a nanostructured lipid carrier and one or more antigens; the antigen is ovalbumin (OVA) or a viral antigen, such as the E2 protein of bovine viral diarrhea virus (BVDV), as well as swine influenza virus antigen, porcine reproductive and respiratory syndrome virus antigen, porcine circovirus antigen, classical swine fever virus antigen, pseudorabies virus antigen, porcine epidemic diarrhea virus antigen, etc.

[0020] Furthermore, the antiviral agent is effective against swine influenza virus H3N2, which can reduce viral load, inhibit cytopathic effects, and alleviate lung inflammation and damage.

[0021] Furthermore, the nanostructured lipid carrier suspension can be directly mixed with the antigen solution, or it can be further mixed with 10% sucrose as a freeze-drying protectant and then freeze-dried to produce a freeze-dried powder formulation. After reconstitution, the freeze-dried powder still maintains good physical stability and immunomodulatory activity, which can extend the shelf life.

[0022] Preparation Principle: This invention utilizes a combination of palmitic acid (solid lipid) and squalene (liquid lipid) to construct the framework of a nanostructured lipid carrier. Palmitic acid helps form a stable crystal structure, while squalene not only serves as an immunomodulatory component but also increases the carrier's solubility for antigens and drugs. Tilololon, as a TLR7 / 8 agonist, is efficiently encapsulated within the lipid core. A special 'ice-water bath assisted high-shear cooling' process promotes rapid lipid solidification, physically encapsulating tilololon internally to form uniformly sized and stably dispersed nanoparticles. This structure exhibits excellent encapsulation and sustained-release capabilities for tilololon and antigens, allowing for the slow release of active ingredients in vivo, synergistically activating innate immunity and enhancing adaptive immune responses.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0024] 1. It has excellent drug loading and sustained-release properties, with an encapsulation rate of up to 89% for telotron, and can achieve slow drug release, prolonging its duration of action in vivo.

[0025] 2. This invention can not only serve as an antigen carrier, but the telotron it encapsulates can also effectively activate immune pathways and synergistically enhance the body's humoral and cellular immune responses, thus exhibiting a good immune-enhancing effect.

[0026] 3. Animal experiments show that the system is safe and stable, and can be further prepared into freeze-dried powder for easy storage and transportation. Attached Figure Description

[0027] Figure 1 This is a particle size distribution diagram of tsNLCs prepared in Example 1 of the present invention;

[0028] Figure 2 This is a Zeta potential distribution map of tsNLCs prepared in Example 1 of the present invention;

[0029] Figure 3 These are transmission electron microscope images of tsNLCs prepared in Example 1 of this invention, wherein A is a 100x magnified image (scale bar = 1 μm); B is a 200x magnified image (scale bar = 500 nm).

[0030] Figure 4 This is a comparison of the in vitro release curves of the tsNLCs of this invention and tilolone active pharmaceutical ingredient;

[0031] Figure 5 This is a graph showing the serum OVA-specific IgG antibody levels in mice after vaccination with an OVA vaccine containing tsNLCs as an adjuvant. In the graph, A represents the significant difference compared to the OVA-tsNLCs group, and B represents the significant difference compared to each time point within the group.

[0032] Figure 6 This is a graph showing the changes in body weight of mice during immunization after receiving the E2 vaccine with tsNLCs as an adjuvant.

[0033] Figure 7 This is a graph showing the serum E2-specific IgG antibody levels in mice after vaccination with the E2 vaccine using tsNLCs as an adjuvant. In the graph, A represents the significant difference compared to the E2-tsNLCs group, and B represents the significant difference compared to each time point within the group.

[0034] Figure 8 CD3+ cells in spleen lymphocytes of mice after vaccination with E2 vaccine using tsNLCs as adjuvants + CD4 + A graph showing changes in lymphocyte subsets, where a represents the CD3+ subsets in the blank control group. + CD4 + Splenic lymphocyte ratio, b represents CD3 in group E2. + CD4 + Splenic lymphocyte ratio, c represents CD3 in the E2-TIL group + CD4 + Splenic lymphocyte ratio, d represents the CD3+CD4+ splenic lymphocyte ratio in the E2-sNLCs group, e represents the CD3+CD4+ splenic lymphocyte ratio in the E2-ISA15A group. + CD4 + Splenic lymphocyte ratio, f represents CD3 in the E2-tsNLCs group + CD4 + Splenic lymphocyte ratio;

[0035] Figure 9CD3+ cells in spleen lymphocytes of mice after vaccination with E2 vaccine using tsNLCs as adjuvants + CD8 + A graph showing changes in lymphocyte subsets, where a represents the CD3+ subsets in the blank control group. + CD8 + Splenic lymphocyte ratio, b represents CD3 in group E2. + CD8 + Splenic lymphocyte ratio, c represents CD3 in the E2-TIL group + CD8 + Splenic lymphocyte ratio, d represents the proportion of CD3+CD8+ splenic lymphocytes in the E2-sNLCs group, e represents the proportion of CD3+CD8+ splenic lymphocytes in the E2-ISA15A group. + CD8 + Splenic lymphocyte ratio, f represents CD3 in the E2-tsNLCs group + CD8 + Splenic lymphocyte ratio;

[0036] Figure 10 The CD3 counts in mice after vaccination with the E2 vaccine using tsNLCs as adjuvants are... + CD4 + A schematic diagram illustrating the significant differences between lymphocyte subsets and the E2-tsNLCs group;

[0037] Figure 11 The CD3 counts in mice after vaccination with the E2 vaccine using tsNLCs as adjuvants are... + CD8 ++ A schematic diagram illustrating the significant differences between lymphocyte subsets and the E2-tsNLCs group;

[0038] Figure 12 This is a schematic diagram illustrating the inhibitory effect of tiloron on CPE in H3N2 virus-infected 3D4 / 21 cells. In the diagram, A is the blank control group, B is the virus-infected group, and C is the tiloron-treated group.

[0039] Figure 13This diagram illustrates the effects of telotron on lung tissue lesions and viral antigen expression in H3N2 virus-infected piglets. A represents a photograph of the lungs of piglets in the control group, B represents the lungs of piglets in the virus-infected group, C represents the lungs of piglets in the prophylactic treatment group, D represents the lungs of piglets in the treatment group, E represents the HE staining image of the lungs of piglets in the control group, F represents the HE staining image of the lungs of piglets in the virus-infected group, G represents the HE staining image of the lungs of piglets in the prophylactic treatment group, H represents the HE staining image of the lungs of piglets in the treatment group, I represents the H3N2 NP immunohistochemical staining image of the lungs of piglets in the control group, J represents the H3N2 NP immunohistochemical staining image of the lungs of piglets in the virus-infected group, K represents the H3N2 NP immunohistochemical staining image of the lungs of piglets in the prophylactic treatment group, and L represents the H3N2 NP immunohistochemical staining image of the lungs of piglets in the treatment group. Detailed Implementation

[0040] Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available. Experimental methods not specifically described in the embodiments are generally performed under standard conditions or as recommended by the manufacturer.

[0041] Example 1

[0042] Preparation of tsNLCs (tsNLCs)

[0043] Ingredients: palmitic acid 0.1g, squalene 0.9g, emulsifier 0.3g (Tween 80: Span 85 = 1:1), 10 mL of 10 mg / mL telotron solution, and 10 mL of pure water.

[0044] Preparation method: Squalene, palmitic acid, Tween 80, and Span 85 were melted in a constant-temperature magnetically stirred water bath (80℃) and stirred until homogeneous to obtain the oil phase. The prescribed amount of telotron was dissolved in water to prepare a telotron solution, which was placed in another vial and heated in an 80℃ water bath to obtain the aqueous phase. The oil phase was poured into the aqueous phase under magnetic stirring, and after mixing, it was sheared for 5 minutes using a high-shear apparatus (11000 rpm) to obtain a crude emulsion. The crude emulsion was treated with an ultrasonic disruptor (300 W) for 20 minutes, then transferred to an equal volume of ice water for shearing for 1 minute, and cooled in an ice-water bath for 10 minutes to obtain the tsNLCs suspension.

[0045] Characterization results: The average hydrated particle size, polydispersity index (PDI), and zeta potential were determined using a dynamic light scattering particle size analyzer. The results are as follows: Figures 1-2 Particle size 143.50±0.46 nm, PDI 0.15±0.02, Zeta potential -14.10±3.17 mV. Figure 3 Transmission electron microscopy showed that the particles were spherical and evenly distributed.

[0046] Example 2

[0047] Determination of telotron encapsulation efficiency and drug loading in tsNLCs

[0048] The encapsulation efficiency and drug loading of three batches of tsNLCs were determined by ultrafiltration centrifugation combined with ultraviolet spectrophotometry. Based on the standard curve and encapsulation efficiency formula, the average encapsulation efficiency of telotron in tsNLCs was (89.336±0.367)% (mean ± SD, n=3), as shown in Table 1. Based on the standard curve and formula, as shown in Table 2, the average concentration of telotron in tsNLCs was (5.043±0.062) mg / mL (mean ± SD, n=3), and the drug loading was (7.652±0.093)% (mean ± SD, n=3).

[0049] Table 1 Encapsulation efficiency measurement results

[0050]

[0051] Table 2 Results of drug loading determination

[0052]

[0053] Example 3

[0054] Study on in vitro release characteristics of tsNLCs

[0055] In vitro release assays of tsNLCs were conducted using a dynamic dialysis system. The specific steps were as follows: ① Three aliquots each of tsNLCs containing 34.09 mg telotron and the active pharmaceutical ingredient were precisely pipetted into dialysis bags. The ends of the dialysis bags were clamped and leak-checked before use. ② Six 500 mL beakers were prepared, each containing 500 mL of release medium (PBS was selected as the release medium). The dialysis bags were placed in the 500 mL beakers, sealed with plastic wrap, and placed in a constant-temperature, digitally displayed magnetically stirred tank. ③ Condition settings: constant temperature 37℃, stirring frequency 120 rpm. ④ Sampling time: 1 mL samples were taken at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 12 h, and an equal volume of fresh release medium was quickly added. ⑤ Sample analysis: The samples were diluted 7.5 times with methanol. Each sample was analyzed using a UV spectrophotometer, and the absorbance at the maximum absorption wavelength was recorded. The drug concentration in the release medium was calculated based on the standard curve equation. ⑥ Calculate the cumulative release of tiloron according to the formula.

[0056] The results are as follows Figure 4As shown, the release rates of tsNLCs and tololon API were significantly different at 0.5 h and extremely significantly different within 1–12 h. Furthermore, the cumulative release rates of tsNLCs and tololon API within 12 h were (49.43±0.21)% and (90.14±1.41)%, respectively. This indicates that tsNLCs have a sustained-release effect, which is beneficial for maintaining a long-lasting immunostimulatory effect.

[0057] Example 4

[0058] Preparation of ovalbumin-loaded tsNLCs (OVA-tsNLCs)

[0059] The preparation process was the same as in Example 1, except that in the cooling and solidification stage, a solution containing OVA protein was used instead of pure water for dispersion and solidification to obtain an initial suspension of OVA-tsNLCs. The HD, ZP, and PDI of the model antigen OVA loaded onto tsNLCs are shown in Table 3.

[0060] Table 3 HD, ZP, and PDI of OVA-tsNLCs

[0061]

[0062] Example 5

[0063] Preparation of lyophilized tsNLCs loaded with BVDV E2 protein (E2-tsNLCs)

[0064] Table 4 HD, ZP, and PDI of E2-tsNLCs

[0065]

[0066] The preparation process was the same as in Example 1. In the cooling and solidification step, a solution containing BVDV E2 protein was used instead of pure water to obtain an initial suspension of E2-tsNLCs. The E2 protein was then loaded into the tsNLCs. The hydrodynamic diameter (HD), zeta potential (ZP), and polydispersity index (PDI) are shown in Table 4.

[0067] Take 10 mL of the above E2-tsNLCs suspension and mix it thoroughly with an equal volume of lyophilization protectant. Dispense the mixture into 10 mL vials, 2 mL per vial. Pre-freeze at -80°C for 3 hours. Then transfer to a freeze dryer and freeze-dry for 48 hours to obtain E2-tsNLCs lyophilized powder.

[0068] Table 5. Quality evaluation of different E2-tsNLCs lyophilized powders

[0069]

[0070] Note: +: dissolves slowly; ++: dissolves relatively slowly; +++: dissolves relatively quickly; ++++: dissolves rapidly.

[0071] The results in Table 5 show that when 10% sucrose is used as the freeze-drying protectant for E2-tsNLCs freeze-dried powder, its reconstitution effect is good, and HD, ZP and PDI are most similar to E2-tsNLCs before freeze-drying. Therefore, 10% sucrose is the freeze-drying protectant for preparing E2-tsNLCs freeze-dried powder.

[0072] One portion of lyophilized powder was taken and 2 mL of sterile ultrapure water was added. The mixture was gently vortexed for 30 seconds to completely redissolve the powder. The particle size, PDI, and Zeta potential of the redissolved suspension were measured. The results showed that the particle size was 166.53 ± 2.18 nm, the PDI was 0.19 ± 0.03, and the Zeta potential was -15.90 ± 0.55 mV. This indicates that the lyophilization-redissolution process had minimal impact on the particle size and stability of the nanoparticles, and the formulation exhibited good redispersibility.

[0073] Example 6

[0074] Evaluation of tsNLCs as adjuvants to enhance the immunogenicity of OVA

[0075] Animals and grouping: Thirty 6-8 week old female BALB / c mice (SPF grade) were randomly divided into 6 groups of 5 mice each: PBS control group, OVA group, OVA-TIL group, OVA-sNLCs group, OVA-ISA15A group, and OVA-tsNLCs group. The mice were immunized subcutaneously in the back on day 0 and day 14, with each mouse receiving 50 μg of OVA.

[0076] Immunization program: All mice were immunized by subcutaneous injection in the back on day 0 and day 14.

[0077] Sample collection and testing: Blood was collected from the orbital venous plexus on days 14 and 28 after the initial immunization. After standing at room temperature for 30 minutes, the serum was centrifuged at 3000 rpm for 10 minutes and then stored at -20°C.

[0078] ELISA was used to detect serum OVA-specific IgG. Results are as follows: Figure 5 As shown, the antibody titer in the OVA-tsNLCs group was significantly higher than that in the control group (P<0.01), while there was no significant difference compared with the commercial adjuvant group (P>0.05), confirming its excellent humoral immune enhancement effect.

[0079] Example 7

[0080] Evaluation of tsNLCs as adjuvants to enhance the immunogenicity and safety of BVDV E2 protein

[0081] Animals, grouping, and immunization: Grouping was the same as in Example 5, except that the OVA antigen was replaced with BVDV E2 protein, and each mouse received an immunization dose of 50 μg of E2 protein. The immunization schedule was the same as in Example 5 (days 0 and 14).

[0082] Safety evaluation: such as Figure 6 During the experiment, the mice showed no abnormalities, their weight gain was normal, and there were no other abnormalities, indicating good safety.

[0083] Evaluation of immune response: such as Figures 7-11 On day 42, blood samples were collected, and the level of specific IgG antibodies in the E2-tsNLCs group remained significantly higher than that in the control group; flow cytometry analysis of spleen lymphocytes showed that CD3 + CD4 + and CD3 + CD8 + The proportion of T cells was significantly increased. These results indicate that tsNLCs can significantly enhance E2 protein-induced humoral and cellular immune responses, demonstrating a good immune adjuvant effect.

[0084] Example 8

[0085] Protective effect of tsNLCs on H3N2 virus-infected cells

[0086] To verify the antiviral function of the tsNLCs prepared in this invention, this embodiment investigated the in vitro inhibitory effect of tsNLCs on swine influenza virus H3N2 infection of 3D4 / 21 cells.

[0087] 3D4 / 21 cells were treated with 100 TCID. 50 Two hours after infection with H3N2 virus solution, telotron was added and the culture continued for another 24 hours. The results were observed under a microscope as follows. Figure 12 As shown, the virus-infected group showed obvious cytopathic effects (CPE), manifested as cell shrinkage, rounding, and shedding; the cells in the telolonol group had intact morphology, and CPE was significantly inhibited.

[0088] Example 9

[0089] Protective effect of tsNLCs on H3N2 virus-infected piglets

[0090] To verify the antiviral function of the tsNLCs prepared in this invention in vivo, this embodiment investigated the protective effect of tsNLCs against swine influenza virus H3N2 infection in piglets.

[0091] Twenty 4-week-old weaned piglets were randomly divided into four groups: a blank control group, a virus infection group, a prophylactic administration group, and a treatment group, with five piglets in each group. Piglets in the virus infection group, prophylactic administration group, and treatment group were inoculated intranasally with 2 mL of H3N2 virus solution, while the blank control group was instilled with an equal volume of sterile PBS. The prophylactic administration group received telotron (10 mg / kg) via gavage daily on days 1 before infection and on days 0, 1, 2, and 5 after infection. The treatment group received telotron (10 mg / kg) via gavage daily on days 0, 1, 2, and 5 after infection. Animals were dissected on day 6 post-infection, and lung tissue was collected for HE staining and immunohistochemical analysis.

[0092] Piglets in the blank control group, prophylactic administration group, and treatment administration group had normal feed and water intake and did not develop fever during the experiment. Piglets in the virus-infected group developed fever and lethargy on day 2 after viral challenge, with a maximum body temperature reaching 41°C. Necropsy results are as follows... Figure 13 As shown, the lung tissue of piglets in the virus-infected group exhibited significant pathological damage, manifested as focal or patchy purplish-red lesions, with congestion, hemorrhage, and consolidation in some lung lobes. HE staining revealed a large number of inflammatory cells aggregated in the alveolar septa and alveolar cavities, with significantly widened alveolar walls, and some alveoli fused or collapsed. The pathological damage to the lungs was significantly reduced, with no obvious edema, inflammatory cell infiltration, or fibrous connective tissue hyperplasia observed, and the alveolar structure remained relatively intact. Immunohistochemical results showed yellow punctate positive signals in the lung tissue of the virus-infected group, indicating the presence of influenza virus antigens; no obvious viral antigen signals were observed in the prophylactic and treatment groups.

[0093] The above results indicate that tsNLCs have significant in vitro and in vivo inhibitory effects on swine influenza virus H3N2, and can effectively reduce virus-induced lung damage, further suggesting that the tsNLCs described in this invention have good application potential in the field of antiviral vaccine adjuvants.

[0094] Example 10

[0095] Preparation of tsNLCs (tsNLCs)

[0096] Ingredients: palmitic acid 0.1g, squalene 0.9g, emulsifier 0.3g (Tween 80: Span 85 = 1:1), 10 mL of 10 mg / mL telotron solution, and 9 mL of pure water.

[0097] Preparation method: Squalene, palmitic acid, Tween 80, and Span 85 were melted in a constant-temperature magnetically stirred water bath (75℃) and stirred until homogeneous to obtain the oil phase. The prescribed amount of telotron was dissolved in water to prepare a telotron solution, which was placed in another vial and heated in an 80℃ water bath to obtain the aqueous phase. The oil phase was poured into the aqueous phase under magnetic stirring, and after mixing, it was sheared for 10 minutes using a high-shear apparatus (10000 rpm) to obtain a crude emulsion. The crude emulsion was treated with an ultrasonic disruptor (200 W) for 25 minutes, then transferred to an equal volume of ice water for shearing for 1 minute, and cooled in an ice-water bath for 10 minutes to obtain the tsNLCs suspension.

[0098] Example 11

[0099] Preparation of tsNLCs (tsNLCs)

[0100] Ingredients: palmitic acid 0.1g, squalene 0.9g, emulsifier 0.3g (Tween 80: Span 85 = 1:1), 10 mL of 10 mg / mL telotron solution, and 9.5 mL of pure water.

[0101] Preparation method: Squalene, palmitic acid, Tween 80, and Span 85 were melted in a constant-temperature magnetically stirred water bath (85℃) and stirred until homogeneous to obtain the oil phase. The prescribed amount of telotron was dissolved in water to prepare a telotron solution, which was placed in another vial and heated in an 80℃ water bath to obtain the aqueous phase. The oil phase was poured into the aqueous phase under magnetic stirring, and after mixing, it was sheared for 3 minutes using a high-shear apparatus (15000 rpm) to obtain a crude emulsion. The crude emulsion was treated with an ultrasonic disruptor (400 W) for 15 minutes, then transferred to an equal volume of ice water for shearing for 1 minute, and cooled in an ice-water bath for 10 minutes to obtain the tsNLCs suspension.

[0102] Comparative Example 1

[0103] The remaining steps of this comparative example are the same as those in Example 1, except that palmitic acid is replaced with stearic acid. Stearic acid has a higher crystallinity than palmitic acid, and it is easier to form a highly ordered crystal structure during the formation of nanolipid carriers, resulting in: (1) telotron being expelled from the lipid lattice, reducing the encapsulation efficiency; (2) easy aggregation between particles, resulting in a wider particle size distribution; (3) drug adsorption onto the particle surface, causing burst release. This shows that palmitic acid, due to its moderate melting point and crystallization characteristics, is the best choice for this invention.

[0104] Comparative Example 2

[0105] The remaining steps of this comparative example are the same as those in Example 1, except that squalene is replaced with medium-chain triglycerides. Squalene not only acts as a liquid lipid to regulate the crystal defects of nanostructured lipid carriers, but more importantly, it has immunoadjuvant activity. After being replaced with medium-chain triglycerides: (1) the carrier's immune-enhancing effect on the antigen is basically lost; (2) it cannot form a synergistic effect with telotron. This shows that squalene is both a structural component and a functional component, and cannot be replaced.

[0106] Comparative Example 3

[0107] The remaining steps of this comparative example are the same as those in Example 1, except that telotron is dissolved in the heated oil phase instead of the aqueous phase. That is, telotron, squalene, palmitic acid, and emulsifier are melted and mixed together in an 80°C water bath. Telotron is highly hydrophilic, and its solubility in the oil phase is much lower than that in the aqueous phase. Adding telotron to the oil phase will result in: (1) most of the drug cannot be effectively encapsulated, resulting in a very low encapsulation rate; (2) the unencapsulated free drug is prone to crystallization in the system. The present invention uses a process in which telotron is dissolved in the aqueous phase, utilizing its distribution behavior at the oil-water interface to achieve efficient encapsulation, which is the key to obtaining a high encapsulation rate.

[0108] Comparative Example 4

[0109] The remaining steps of this comparative example are the same as those in Example 1, except that step four is replaced with "slowly stirring the ultrasonically treated nanoemulsion at room temperature and naturally cooling it to room temperature to obtain a nanostructured lipid carrier suspension". The key to this invention is "ice-water bath assisted high-shear cooling" to rapidly solidify the lipids and physically encapsulate telotron in the lipid lattice defects. During natural cooling: (1) the lipid crystallization rate is slow, and larger particles are easily formed; (2) the drug is squeezed out during slow cooling, resulting in a decrease in encapsulation efficiency and burst release; (3) particles are prone to fusion and aggregation. This shows that the ice-water bath rapid cooling process is a key technical feature for controlling particle size and ensuring encapsulation efficiency and stability.

[0110] Comparative Example 5

[0111] The remaining steps of this comparative example are the same as those in Example 1, except that the ultrasonic treatment time in step three is changed from 20 minutes to 5 minutes. The key function of ultrasonic treatment is to further break down the crude emulsion into nanoscale particles. Too short a time means insufficient energy input, resulting in: (1) large particles are not fully broken down, resulting in larger particle sizes; (2) the particle size distribution is wide, making it easy for stratification or sedimentation to occur. The preferred ultrasonic time of 20 minutes in Example 1 of this invention ensures sufficient energy input and obtains a nano-formulation with uniform and stable particle size.

[0112] Of the above embodiments, Embodiment 1 is the optimal embodiment.

Claims

1. A method for preparing a tilaurolon nanostructured lipid carrier, characterized in that, Includes the following steps: Step 1: Heat and melt the solid lipids, squalene, and emulsifier to form an oil phase, and dissolve tilaron in heated water to form an aqueous phase; Step 2: Mix the oil phase and the water phase, and perform high-shear emulsification to obtain a crude emulsion; Step 3: The crude emulsion is subjected to ultrasonic treatment to obtain a nanoemulsion; Step four: The nanoemulsion is transferred to ice water for shearing and cooled in an ice water bath to obtain a nanostructured lipid carrier.

2. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step one, the mass ratio of solid lipids, squalene, emulsifier, telotron, and water is 1:9:3:1:90~100.

3. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step one, the solid lipid is palmitic acid.

4. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step one, the emulsifiers are equal masses of Tween 80 and Span 85.

5. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step one, the heating temperature is 75~85℃.

6. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step two, the high-shear emulsification speed is 10,000 to 12,000 rpm, and the time is 3 to 10 minutes.

7. The method for preparing a nanostructured lipid carrier according to claim 1, characterized in that: In step three, the ultrasonic treatment power is 200~400 W and the time is 15~25 minutes.

8. A nanostructured lipid carrier prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The average hydrated particle size of the nanostructured lipid carrier is 100~200 nm, the polydispersity index is less than 0.25, and the zeta potential is -10 ~ -20 mV.

9. The use of the nanostructured lipid carrier of claim 8 in the preparation of a vaccine composition.

10. The application according to claim 9, characterized in that: The vaccine composition includes a nanostructured lipid carrier and one or more antigens; the antigen is ovalbumin or a viral antigen.

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