MPL composite adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, and discloses an MPL composite adjuvant which comprises the following components in parts by mass: 10-20 parts of MPL; 5 to 15 parts by mass of immunomodulatory peptide; the mass part of the nano carrier is 20 to 40 parts; 1-5 parts by mass of an auxiliary stable component; the preparation method of the MPL composite adjuvant comprises the following steps: dissolving MPL in an organic solvent to obtain an MPL solution, dissolving immunomodulatory peptide in a buffer solution, adding a nano-carrier solution into the mixed solution, filtering the mixed solution, and converting the mixed solution into a solid form through freeze drying, the MPL composite adjuvant is applied to vaccine development. According to the invention, a nano-carrier technology of combining lecithin and vitamin E is adopted, so that the stability and immune activation capability of the MPL composite adjuvant are improved, and the immune effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to an MPL composite adjuvant and a preparation method and application thereof. Background Art

[0002] Immune adjuvants play an important role in medicine, vaccine development and immunotherapy. With the development of biotechnology, the research on immune adjuvants has evolved from simple immune enhancement to more complex stability and durability optimization. We hope to improve the formula of immune adjuvants so that they can not only effectively enhance the immune response, but also work for a long time in the body. Especially in vaccines and immunotherapy, adjuvants can help activate the immune system and improve the immune effect against pathogens, thereby achieving better protection.

[0003] In the prior art, common methods of immune adjuvants mainly rely on a single carrier material, such as lecithin or phosphatidylcholine, which can enhance the immune response to a certain extent and promote the recognition of the immune system. However, these technologies are limited in the stability of adjuvants. In order to improve the immune effect of adjuvants, some technologies also regulate the particle size and select the appropriate nanoparticle size to promote the uptake and response of immune cells. In addition, some technologies also use freeze-drying methods to improve the preservation effect of adjuvants, thereby reducing the degradation of components during storage and transportation and ensuring the long-term stability of immune adjuvants.

[0004] However, the prior art still has obvious deficiencies in the stability and immune effect of immune adjuvants. First, adjuvants made of a single carrier material are easily affected by environmental factors and are easily degraded during long-term storage, resulting in fluctuations in immune effect. Second, although nanocarriers with larger particle sizes can prolong the residence time of adjuvants in the body, the immune response is slow to initiate and the effect is difficult to sustain. Moreover, most of the prior art ignores surface functionalization treatment, resulting in the adjuvant being cleared too quickly in the immune system and failing to continuously exert the desired immune effect. In addition, although freeze-drying technology can improve the storage stability of adjuvants, it still cannot effectively solve the problem of insufficient adjuvant immune activation ability. For this reason, those skilled in the art propose an MPL composite adjuvant and its preparation method and application to solve the above problems. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an MPL composite adjuvant and a preparation method and application thereof, which solves the problems of poor stability of immune adjuvants, unsustainable immune effects, imprecise particle size control and insufficient surface functionalization treatment in the prior art.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: an MPL composite adjuvant, comprising the following components: MPL, mass parts are 10-20 parts; Immunomodulatory peptide, 5-15 parts by weight; Nanocarrier, 20-40 parts by weight; Auxiliary stabilizing component, mass fraction is 1-5 parts.

[0007] The MPL composite adjuvant uses MPL as the main immunogen, and significantly enhances the immunostimulatory effect of the adjuvant by combining it with immunomodulatory peptides and nanocarriers. As a TLR4 receptor agonist, MPL can stimulate the host's immune system, promote the activation of immune cells and inflammatory responses, thereby enhancing the immune response of the vaccine. Through the synergistic effect with MPL, immunomodulatory peptides further optimize the activation process of the immune system and enhance the intensity and specificity of the immune response. The use of nanocarriers not only improves the stability of the adjuvant, but also enables the adjuvant to be precisely delivered to the target site through its excellent drug delivery properties, further enhancing the immune effect. The addition of auxiliary stabilizing ingredients such as antioxidants can effectively protect the active ingredients in the adjuvant from oxidative damage, thereby ensuring its long-term stability.

[0008] Preferably, the immunomodulatory peptide is a TLR4 agonist peptide; The nanocarrier is a lipid nanoparticle, and the lipid is soybean lecithin or a lecithin component; The stabilizing component is an antioxidant, and the antioxidant is selected from vitamin C, vitamin E or glutathione.

[0009] TLR4 receptor agonist peptides can enhance the host's immune response and promote the activation of the immune system by specifically binding to the TLR4 receptor, especially playing a key role in antigen presentation and T cell activation. Lipid nanoparticles, as nanocarriers, are composed of soybean lecithin or lecithin components, have good biocompatibility and strong drug delivery capabilities, and can effectively encapsulate and deliver MPL and immunomodulatory peptides, thereby reducing drug degradation and nonspecific distribution, and improving drug targeting and bioavailability. Antioxidants such as vitamin C, vitamin E or glutathione can prevent oxidation reactions of adjuvants during storage and transportation, maintain the stability of adjuvants, and avoid side effects caused by oxidation, thereby ensuring the long-term effectiveness of adjuvants.

[0010] A method for preparing an MPL composite adjuvant comprises the following steps: Dissolving MPL in an organic solvent to obtain an MPL solution, wherein the solvent is ethanol or dichloromethane; Dissolve the immunomodulatory peptide in the buffer solution and add it to the MPL solution and stir evenly; The binding of MPL to immunomodulatory peptides was optimized by adjusting the pH value to 4.5-7.5 and the ionic strength to 0.1-1 mol / L; Adding the nanocarrier solution to the mixed solution, and stirring to allow the MPL and the immunomodulatory peptide to be fully encapsulated in the nanocarrier; Adding auxiliary stabilizing components to the mixed solution and stirring evenly to enhance the stability of the adjuvant; Filtering the mixed solution; The mixed solution is converted into a solid form by freeze drying to ensure the stability of the composite adjuvant.

[0011] First, after MPL is dissolved in an organic solvent, immunomodulatory peptides are added and the pH and ionic strength are adjusted to optimize the binding of MPL and immunomodulatory peptides. This step is key to ensuring that MPL and immunomodulatory peptides can form a stable complex. The optimized pH and ionic strength help to improve the binding efficiency between the two, thereby improving the immunostimulatory effect. The introduction of nanocarriers is to encapsulate MPL and immunomodulatory peptides to ensure their stability and effective delivery in the body. After adding auxiliary stabilizing ingredients, the stability of the adjuvant is further improved to prevent the adjuvant from degrading during storage and use. Finally, the adjuvant is converted into a solid by freeze-drying, which is convenient for long-term storage and transportation while maintaining the activity of the adjuvant.

[0012] Preferably, the dissolution time of the MPL solution is 30-60 min, the dissolution temperature is 25-30° C., and the stirring rate is 300-600 rpm.

[0013] The conditions for dissolving MPL are crucial to the quality of the final adjuvant. By controlling the dissolution time, dissolution temperature and stirring rate, the complete dissolution of MPL can be ensured, and aggregation or degradation of MPL during the dissolution process can be avoided. Appropriate dissolution conditions not only improve the solubility of MPL, but also ensure its activity in subsequent reactions, providing a stable material basis for subsequent immune stimulation.

[0014] Preferably, the dissolution time of the immunomodulatory peptide is 20-30 min, the dissolution temperature is 25-30° C., and the stirring rate is 200-300 rpm.

[0015] The dissolution process of the immunomodulatory peptide also needs to be strictly controlled to ensure that it does not degrade or aggregate during the dissolution process. By controlling the dissolution time, temperature and stirring rate, the stability and activity of the immunomodulatory peptide can be effectively guaranteed, thereby ensuring its effect when combined with MPL. These control conditions can ensure that the immunomodulatory peptide achieves the best reaction efficiency when combined with MPL, further enhancing the immune effect of the adjuvant.

[0016] Preferably, the preparation method of the nanocarrier comprises: The lipid is dissolved in a solvent, and the solvent is removed by rotary evaporation to obtain a nanocarrier, and the nanocarrier is subjected to surface functionalization treatment. The particle size of the nanocarrier is in the range of 50-200 nm.

[0017] By selecting soybean lecithin or egg choline as the main component of the nanocarrier and removing the solvent by rotary evaporation, lipid nanoparticles with uniform size and good stability can be obtained. The good biocompatibility and biodegradability of phospholipids enable this nanocarrier to not only encapsulate MPL and immunomodulatory peptides, but also achieve a sustained release effect in vivo, thereby enhancing the effect of the adjuvant. The selection of the nanocarrier particle size range (50-200nm) ensures the stability and targeting of the carrier in vivo, avoiding immune tolerance caused by overly large particles or short-term elimination of overly small particles.

[0018] Preferably, the surface functionalization treatment of the nanocarrier comprises the following steps: The nanocarrier is treated with a PEGylation reagent, wherein the mass of the PEGylation reagent accounts for 0.1%-1.0% of the mass of the nanocarrier, the reaction temperature is 30-40° C., and the reaction time is 2-4 hours.

[0019] PEG (polyethylene glycol) is a commonly used biocompatibility enhancer that can significantly improve the biostability of nanocarriers, reduce recognition and clearance by the body's immune system, and thus prolong their half-life in the body. PEGylation allows nanocarriers to remain stable in the blood for a longer period of time, and reduces nonspecific binding to plasma proteins, avoiding clearance by the immune system. At the same time, PEGylation can also enhance the hydrophilicity of nanocarriers, improve their dispersibility in aqueous solutions, and further improve the delivery efficiency and bioavailability of composite adjuvants.

[0020] Preferably, the filtering step comprises: Use a 0.22 μm filter membrane for pre-filtration to remove larger impurities and ensure the initial purity of the solution; Then, ultrafiltration with a pore size range of 0.05-0.2 μm is used to further remove tiny particles and microorganisms to ensure the purity and sterility of the composite adjuvant.

[0021] Filtration and ultrafiltration are necessary steps to ensure the quality of composite adjuvants. Pre-filtration using a 0.22μm filter membrane can effectively remove larger particles, impurities and potential contaminants in the solution. This process helps to initially improve the purity of the solution. Subsequently, through ultrafiltration treatment, an ultrafiltration membrane with a pore size range of 0.05-0.2μm can effectively remove bacteria, viruses, and other tiny particles in the solution to ensure the sterility and purity of the composite adjuvant. Ultrafiltration can not only remove potential microbial contamination, but also further remove some soluble impurities, thereby ensuring the quality and safety of the final product.

[0022] Preferably, the composite adjuvant solution is frozen at -80°C for 2 hours, and then dried using a freeze-drying device for 12 hours.

[0023] The freeze-drying (lyophilization) process is a key step to ensure the stability of the composite adjuvant during long-term storage. By freezing the composite adjuvant solution at -80°C and keeping it for a certain period of time, the water activity of the adjuvant solution can be quickly reduced, inhibiting its degradation or deterioration. At the same time, the use of freeze-drying equipment can remove excess water to form a stable solid powder form for easy storage and transportation. The freeze-dried adjuvant can still maintain its activity and is easy to re-dissolve and use when in use. Through this treatment method, the stability of the composite adjuvant is greatly improved, especially the protective effect on components such as antigens and immunomodulatory peptides.

[0024] Application of an MPL composite adjuvant in vaccine development.

[0025] The composite adjuvant of the present invention has the ability to significantly enhance the immune response by optimizing its formula and preparation process. In vaccine development, the MPL composite adjuvant can be used as an effective immunopotentiator to enhance the strength and specificity of the immune response of the vaccine. Through the synergistic effect with immunomodulatory peptides and nanocarriers, the adjuvant of the present invention can increase the antibody level after vaccination, enhance the immune memory to the antigen, and reduce the side effects caused by traditional adjuvants. The targeted delivery characteristics of nanocarriers ensure that the immunostimulants can reach the immune cells more effectively, optimize the effect of the immune response, and thus improve the safety and effectiveness of the vaccine.

[0026] The present invention provides an MPL composite adjuvant and a preparation method and application thereof, which have the following beneficial effects: 1. The present invention adopts nanocarrier technology combining lecithin and vitamin E to improve the stability and immune activation ability of the MPL composite adjuvant. Compared with the solution using a single carrier material in the prior art, this combination not only effectively reduces the degradation of the adjuvant, but also enhances its sustained effect in the body and improves the immune effect.

[0027] 2. The present invention optimizes the distribution of adjuvants and the recognition ability of the immune system by regulating the particle size range of nanocarriers to be between 100-130nm. Compared with the traditional scheme of using larger particle size carriers, the particle size regulation of the present invention improves the efficiency of the immune response and avoids the problem of delayed immune response caused by excessively large particle size.

[0028] 3. The present invention introduces freeze-drying technology to process adjuvants, which improves the stability and immune effect of adjuvants during long-term storage. Compared with conventional liquid adjuvant storage methods, this technical solution solves the problem of degradation of active ingredients during storage and ensures the high efficiency of adjuvants during transportation and storage.

[0029] 4. The present invention adopts a combination of multiple surface functionalization treatments and auxiliary stabilizing components to improve the immune response ability of the MPL composite adjuvant. Compared with the traditional technical solution that only relies on a single stabilizer, the additional functionalization treatment enables the adjuvant to exhibit a more lasting and efficient immune activation effect when facing different immune challenges. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Please see attached Figure 1 .

[0033] Embodiment 1: Components: MPL: 15 copies; Immunomodulatory peptide: 10 copies (TLR4 agonist peptide); Nanocarrier: 30 parts (soy lecithin, lipid nanoparticles); Auxiliary stabilizing ingredients: 2 parts (Vitamin C).

[0034] 1. MPL dissolution and immunomodulatory peptide dissolution: 15 parts of MPL were dissolved in 30 parts of dichloromethane solvent and stirred at 28°C for 45 min to ensure that MPL was completely dissolved.

[0035] 10 portions of TLR4 agonist peptide were dissolved in 1×PBS buffer to obtain an immunomodulatory peptide solution. The dissolution time was 30 min and the stirring rate was 300 rpm.

[0036] 2. MPL binds to immunomodulatory peptides: Slowly add the immunomodulatory peptide solution into the MPL solution, adjust the pH value to 6.5, keep stirring to ensure that MPL and immunomodulatory peptide are fully combined, and the stirring time is 30 minutes.

[0037] 3. Preparation and mixing of nanocarriers: 30 parts of soybean lecithin were dissolved in chloroform, and the solvent was removed by rotary evaporation to obtain a lipid film.

[0038] The lipid film was redissolved in an appropriate amount of 1×PBS buffer to form lipid nanoparticles. The nanoparticles were crushed by ultrasound to obtain a particle size of 120 nm.

[0039] The nanocarrier solution was added to the mixed solution of MPL and immunomodulatory peptide, and stirred at 300 rpm for 2 h.

[0040] 4. Addition and mixing of auxiliary stabilizing ingredients: Add 2 parts of vitamin C to the above mixed solution and continue stirring for 30 minutes to ensure uniform distribution to enhance the stability of the adjuvant.

[0041] 5. Filtration and drying: The mixed solution was pre-filtered using a 0.22 μm filter membrane to remove larger impurities.

[0042] The product is further processed using a 0.05 μm ultrafiltration membrane to remove tiny particles and microorganisms.

[0043] The filtered solution was frozen at -80°C for 2 h, and dried by freeze drying equipment for 12 h to obtain the final MPL composite adjuvant powder.

[0044] Embodiment 2: Components: MPL: 18 copies; Immunomodulatory peptide: 8 copies (TLR4 agonist peptide); Nanocarrier: 35 parts (lecithin, lipid nanoparticles); Auxiliary stabilizing ingredient: 3 parts (glutathione).

[0045] 1. MPL dissolution and immunomodulatory peptide dissolution: 18 parts of MPL were dissolved in 35 parts of ethanol and stirred at 25°C for 60 min to ensure that MPL was completely dissolved.

[0046] Eight portions of TLR4 agonist peptide were dissolved in 1×PBS buffer. The dissolution time was 30 min and the stirring rate was 400 rpm.

[0047] 2. MPL binds to immunomodulatory peptides: Slowly add the immunomodulatory peptide solution to the MPL solution, adjust the pH to 6.8, and keep stirring to ensure full combination. The stirring time is 40 minutes.

[0048] 3. Preparation and mixing of nanocarriers: 35 parts of lecithin were dissolved in chloroform, and the solvent was removed by rotary evaporation to obtain a lipid film.

[0049] The lipid film was redissolved in an appropriate amount of 1×PBS buffer by ultrasonic disruption to form lipid nanoparticles with a particle size range of 100-120 nm.

[0050] The nanocarrier solution was added to the mixed solution of MPL and immunomodulatory peptide, and stirred at 400 rpm for 3 h.

[0051] 4. Addition and mixing of auxiliary stabilizing ingredients: Add 3 parts of glutathione to the mixed solution and stir for 30 min to ensure uniform distribution to enhance the stability of the adjuvant.

[0052] 5. Filtration and drying: The mixed solution was pre-filtered using a 0.22 μm filter membrane.

[0053] Ultrafiltration is used to remove tiny particles and microorganisms using a filter membrane with a pore size of 0.1μm.

[0054] The filtered solution was frozen at -80°C for 2 h, and then dried using a freeze-drying device for 12 h to obtain the final MPL composite adjuvant powder.

[0055] Embodiment 3: Components: MPL: 12 copies; Immunomodulatory peptides: 7 copies (TLR4 agonist peptide); Nanocarrier: 25 parts (soy lecithin, lipid nanoparticles); Auxiliary stabilizing ingredients: 1 part (Vitamin E).

[0056] 1. MPL dissolution and immunomodulatory peptide dissolution: 12 parts of MPL were dissolved in 25 parts of dichloromethane and stirred at 26°C for 30 min to ensure that MPL was completely dissolved.

[0057] Seven portions of TLR4 agonist peptide were dissolved in 1×PBS buffer. The dissolution time was 25 min and the stirring rate was 350 rpm.

[0058] 2. MPL binds to immunomodulatory peptides: The immunomodulatory peptide solution was slowly added to the MPL solution, the pH value was adjusted to 6.3, and stirring was maintained to ensure that MPL and the immunomodulatory peptide were fully combined. The stirring time was 30 min.

[0059] 3. Preparation and mixing of nanocarriers: 25 parts of soybean lecithin was dissolved in chloroform, and the solvent was removed by rotary evaporation to obtain a lipid film.

[0060] The lipid film was redissolved in an appropriate amount of 1×PBS buffer to obtain lipid nanoparticles with a particle size range of 110-130 nm.

[0061] The nanocarrier solution was added to the mixed solution of MPL and immunomodulatory peptide, and stirred at 350 rpm for 2 h.

[0062] 4. Addition and mixing of auxiliary stabilizing ingredients: Add 1 portion of vitamin E to the mixed solution and continue stirring for 30 min to ensure the stability of the adjuvant.

[0063] 5. Filtration and drying: Use a 0.22 μm filter membrane for pre-filtration to remove larger impurities.

[0064] It is then further processed using an ultrafiltration membrane with a pore size of 0.1 μm to remove tiny particles and microorganisms.

[0065] The solution was frozen at -80°C for 2 h and dried using a freeze-drying device for 12 h to obtain the final MPL composite adjuvant powder.

[0066] Comparative Example 1: Compared with Example 1, the difference is that the immunomodulatory peptide used is a TLR2 agonist peptide, and the rest is the same.

[0067] Comparative Example 2: Compared with Example 1, the difference is that the auxiliary stabilizing component is removed and vitamin C is not added, and the rest is the same.

[0068] Comparative Example 3: Compared with Example 2, the difference is that the nanocarrier uses phosphatidylcholine instead of lecithin, and the particle size range is 150-170nm, and the rest is the same.

[0069] Comparative Example 4: Compared with Example 2, the difference is that the dissolution time of the immunomodulatory peptide is 15 min, the dissolution temperature is 20° C., and the stirring rate is 500 rpm, and the rest are the same.

[0070] Comparative Example 5: Compared with Example 3, the difference is that the particle size range of the nanocarrier is 80-100 nm, and the surface PEGylation treatment is not performed, and the rest are the same.

[0071] Comparative Example 6: Compared with Example 3, the difference is that the auxiliary stabilizing ingredient is changed to vitamin E instead of vitamin C, and the freeze-drying time is changed to 16 hours, and the rest is the same.

[0072] Experiment 1: Purpose: Verify the effects of different immunomodulatory peptides and auxiliary stabilizing components on the immune effects of MPL composite adjuvants, especially T cell immune responses and antibody production.

[0073] Experimental Group: Example 1 (TLR4 agonist peptide + vitamin C); Comparative Example 1 (TLR2 agonist peptide + vitamin C); Comparative Example 2 (TLR4 agonist peptide + no auxiliary stabilizing component).

[0074] Experimental steps: 1. Animal grouping and injection: Thirty healthy male C57BL / 6 mice were randomly divided into three groups, with 10 mice in each group. Each group of mice was subcutaneously injected with the corresponding MPL compound adjuvant, with an injection volume of 100 μL per mouse, and the immunization was continued once.

[0075] 2. Immune response detection: One week after immunization, the mouse serum was collected to measure the antibody level. The specific method was to use ELISA to detect the IgG antibody titer of anti-MPL antibodies.

[0076] 3. T cell response assessment: Mouse spleen cells were obtained and the activation of CD4+ and CD8+ T cells was detected by flow cytometry. The secretion levels of IFN-γ and TNF-α were analyzed.

[0077] 4. Cytokine determination: ELISA was used to measure the levels of cytokines such as IFN-γ, TNF-α, and IL-4 to evaluate the type of immune response.

[0078] 5. Data analysis: SPSS software was used for statistical analysis to compare the differences in antibody levels, T cell activation, and cytokine secretion among the groups (the experimental results are shown in Table 1 ).

[0079] Table 1: Experiment 1 immune response results data Experimental Group IgG antibody titer (unit: 1:1000) CD4+T cell activation rate (%) CD8+T cell activation rate (%) IFN-γ level (pg / mL) TNF-α level (pg / mL) IL-4 level (pg / mL) Example 1 3200 42 30 280 250 150 Comparative Example 1 2500 35 27 230 210 180 Comparative Example 2 1500 25 20 180 160 200 Example 1 3300 40 33 290 240 170 Comparative Example 1 2600 33 29 210 220 190 Comparative Example 2 1400 23 19 160 170 210 From Table 1, we can get: The results of this experiment show that different immunomodulatory peptides and auxiliary stabilizing components have a significant effect on the immune effect of the MPL composite adjuvant. The TLR4 agonist peptide used in Example 1 combined with vitamin C significantly increased the antibody level and T cell activation rate of mice. As an important immune receptor, TLR4 can activate the immune system by recognizing lipopolysaccharide (LPS) in the MPL adjuvant, thereby enhancing the immune response. As an antioxidant, vitamin C can not only reduce the negative effects of free radicals on the immune system, but also improve the stability of the adjuvant, thereby enhancing the overall immune effect.

[0080] In Comparative Example 1, TLR2 agonist peptide was used instead of TLR4 agonist peptide. Although it can also activate immune response, the intensity of immune response is weakened compared with Example 1. TLR2 and TLR4 belong to different immune receptors, and their roles in immune activation are different. TLR4 can induce a higher level of immune response through a stronger signal transduction pathway, thereby more effectively activating T cells and promoting antibody production.

[0081] In Comparative Example 2, the auxiliary stabilizing component vitamin C was removed. The experimental results showed that the immune response of this group of mice was significantly lower than that of Example 1. This shows that the addition of vitamin C not only enhances the immune response, but also prevents premature degradation of adjuvant components in the body by improving the stability of the adjuvant, thereby ensuring a more lasting immune effect. This result supports the previously proposed mechanism, namely, the enhancement of the immune effect by the stability of the adjuvant and the auxiliary stabilizing components.

[0082] Experiment 2: Purpose: Verify the effects of different nanocarrier components and processing conditions on the stability and immune effect of MPL composite adjuvant, especially the effects of differences in particle size and surface treatment on the immune response.

[0083] Experimental Group: Example 2 (lecithin + particle size 100-120nm + TLR4 agonist peptide); Comparative Example 3 (phosphatidylcholine + particle size 150-170nm + TLR4 agonist peptide); Comparative Example 4 (lecithin + particle size 100-120nm + TLR4 agonist peptide, immunomodulatory peptide dissolution time 15min, dissolution temperature 20°C, stirring rate 500rpm).

[0084] Experimental steps: 1. Preparation of Nanocarriers: The nanoparticles of three MPL composite adjuvants were prepared by ultrasonic emulsification. For Example 2 and Comparative Example 3, the ratio of lecithin or phosphatidylcholine was adjusted to ensure that the particle size was 100-120nm and 150-170nm, respectively. The carrier of Comparative Example 4 was subjected to an additional immunomodulatory peptide dissolution treatment to ensure that its dissolution time was 15min, and a lower temperature and stirring rate were used.

[0085] 2. Immunization and response assessment: The mice were randomly divided into three groups, 10 in each group, and injected with the three different MPL compound adjuvants mentioned above. Each mouse was injected with 100 μL of the nanocarrier solution, and the immune response was evaluated after vaccination.

[0086] 3. Immune response detection: One week after immunization, mouse sera were collected and the IgG antibody titer of anti-MPL antibodies was measured using the ELISA method.

[0087] At the same time, spleen cells were collected, and the activation of CD4+ and CD8+ T cells was analyzed by flow cytometry, and the levels of cytokines such as IFN-γ, TNF-α, and IL-4 were determined.

[0088] 4. Stability test: Storage stability experiments were conducted on each group of adjuvants to determine the immune effects of the adjuvants after being stored at 4°C and -20°C for 3 months. Dynamic light scattering (DLS) was used to measure the change in particle size and evaluate the dispersibility of the nanocarriers.

[0089] 5. Data analysis: SPSS software was used to perform statistical analysis on the experimental data and compare the immune responses and adjuvant stability of each group, especially the effects of particle size and surface treatment on the immune effect (the experimental results are shown in Table 2).

[0090] Table 2: Experiment 2 immune response and stability test results Experimental Group IgG antibody titer (unit: 1:1000) CD4+T cell activation rate (%) CD8+T cell activation rate (%) IFN-γ level (pg / mL) TNF-α level (pg / mL) IL-4 level (pg / mL) Example 2 3500 45 36 300 280 170 Comparative Example 3 2900 38 32 260 240 160 Comparative Example 4 3200 42 35 280 260 180 Example 2 3400 47 39 310 290 175 Comparative Example 3 2800 34 28 250 230 165 Comparative Example 4 3100 41 34 270 250 185 From Table 2, we can get: In this experiment, we observed the significant effects of different nanocarrier components and particle sizes on the immune effect of MPL composite adjuvant. The lecithin used in Example 2 as a carrier, with a particle size in the range of 100-120nm, showed a relatively high level of immune response. According to the mechanism analysis, a smaller particle size is conducive to better recognition and uptake of the adjuvant by the immune system, which can promote the distribution and duration of the adjuvant in the body, thereby enhancing the immune response.

[0091] In contrast, the phosphatidylcholine and larger particle size (150-170nm) used in Comparative Example 3 resulted in a weaker immune response. This result is consistent with the mechanism of the effect of particle size on immune response. A larger particle size may slow down the speed at which nanoparticles are taken up by immune cells such as dendritic cells, thereby reducing their efficiency in activating immune responses. Adjuvants with larger particle sizes may stay in the body for a longer time, but their immune activation ability is weaker.

[0092] The adjuvant in Comparative Example 4 used a lecithin carrier with the same particle size, but by adjusting the time and conditions for dissolving the immunomodulatory peptide, the results showed that the intensity of its immune response was improved. This shows that appropriate dissolution time and temperature conditions can improve the stability of the adjuvant and optimize its immune effect. This finding shows that in addition to particle size and carrier type, minor adjustments in processing conditions during adjuvant preparation may also significantly affect the final immune response intensity.

[0093] Experiment 3: Purpose: Verify the effects of different particle sizes, surface functionalization treatments and auxiliary stabilizing components on the immune effect and stability of MPL composite adjuvants, especially the effects of differences in immunomodulatory peptide solubility and surface treatment on the immune response.

[0094] Experimental Group: Example 3 (soy lecithin + particle size 110-130 nm + vitamin E); Comparative Example 5 (soy lecithin + particle size 80-100 nm + no surface PEGylation treatment); Comparative Example 6 (soy lecithin + particle size 110-130 nm + vitamin E, freeze-drying time 16 hours).

[0095] Experimental steps: 1. Preparation of Nanocarriers: The three MPL composite adjuvant nanocarriers were prepared by ultrasonic emulsification, and the particle sizes were 110-130 nm (experimental groups 1 and 3) and 80-100 nm (experimental group 2). The carriers of experimental group 3 were freeze-dried, and the freeze-drying time was 16 h.

[0096] 2. Immunization and response assessment: The mice were randomly divided into three groups, 10 in each group, and injected with the three different MPL compound adjuvants mentioned above. Each mouse was injected with 100 μL of the nanocarrier solution, and the immune response was evaluated after injection.

[0097] 3. Immune response detection: One week after immunization, mouse sera were collected and the IgG antibody titer of anti-MPL antibodies was measured using the ELISA method.

[0098] At the same time, spleen cells were collected, and the activation of CD4+ and CD8+ T cells was analyzed by flow cytometry, and the levels of cytokines such as IFN-γ, TNF-α, and IL-4 were determined.

[0099] 4. Stability test: Storage stability experiments were conducted on each group of adjuvants to determine the immune effects of the adjuvants after being stored at 4°C and -20°C for 3 months. Dynamic light scattering (DLS) was used to measure the change in particle size and evaluate the dispersibility of the nanocarriers.

[0100] 5. Data analysis: SPSS software was used to perform statistical analysis on the experimental data and compare the immune responses and adjuvant stability of each group, especially the effects of particle size and surface treatment on the immune effect (the experimental results are shown in Table 3).

[0101] Table 3: Experiment 3 immune response and stability test results Experimental Group IgG antibody titer (unit: 1:1000) CD4+T cell activation rate (%) CD8+T cell activation rate (%) IFN-γ level (pg / mL) TNF-α level (pg / mL) IL-4 level (pg / mL) Example 3 3600 50 38 320 300 160 Comparative Example 5 2900 42 30 270 250 175 Comparative Example 6 3300 47 34 310 280 165 Example 3 3500 53 40 330 310 155 Comparative Example 5 2800 38 28 260 240 180 Comparative Example 6 3200 45 33 300 270 170 From Table 3, we can get: In this experiment, we observed that different particle sizes, surface functionalization treatments and auxiliary stabilizing ingredients had a significant effect on the immune effect of the MPL composite adjuvant. Example 3 used soybean lecithin as a carrier with a particle size in the range of 110-130nm, and added vitamin E as an auxiliary stabilizing ingredient, showing a strong immune response. Nanoparticles with moderate particle sizes can be better recognized by the immune system and activate immune responses. Vitamin E, as an antioxidant, enhances the stability of the adjuvant and may reduce the adverse effects of oxidative stress on the immune system during the immunization process.

[0102] Although the smaller particle size (80-100nm) used in Comparative Example 5 is slightly inferior in immune activation, its smaller particle size may increase the rate of its phagocytosis by immune cells such as dendritic cells, thereby accelerating the initiation of immune response. However, the smaller particle size may also cause it to distribute faster in the body, making it difficult to act continuously, and thus the intensity of immune response is lower. In contrast, Comparative Example 6 uses a carrier with the same particle size, but processes the adjuvant through freeze-drying technology to make it more stable during storage and transportation. Freeze-drying can not only reduce the physical degradation of the adjuvant, but also improve its persistence in the body, thereby enhancing the immune effect.

[0103] These results indicate that the immune effect of MPL composite adjuvant is affected by a combination of factors, including particle size, surface treatment, and the addition of auxiliary components. On this basis, we can further optimize the design of adjuvants to enable them to exert greater potential in immunotherapy.

[0104] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A MPL composite adjuvant, characterized in that Includes the following components: MPL, mass parts are 10-20 parts; Immunomodulatory peptide, 5-15 parts by weight; Nanocarrier, 20-40 parts by weight; Auxiliary stabilizing component, mass fraction is 1-5 parts.

2. A MPL composite adjuvant according to claim 1, characterized in that, The immunomodulatory peptide is a TLR4 agonist peptide; The nanocarrier is a lipid nanoparticle, and the lipid is soybean lecithin or a lecithin component; The stabilizing component is an antioxidant, and the antioxidant is selected from vitamin C, vitamin E or glutathione.

3. A method for preparing an MPL composite adjuvant, applied to an MPL composite adjuvant according to claim 1 or 2, characterized in that: The following steps are involved: Dissolving MPL in an organic solvent to obtain an MPL solution, wherein the solvent is ethanol or dichloromethane; Dissolve the immunomodulatory peptide in the buffer solution and add it to the MPL solution and stir evenly; The binding of MPL to immunomodulatory peptides was optimized by adjusting the pH value to 4.5-7.5 and the ionic strength to 0.1-1 mol / L; Adding the nanocarrier solution to the mixed solution, and stirring to allow the MPL and the immunomodulatory peptide to be fully encapsulated in the nanocarrier; Adding auxiliary stabilizing components to the mixed solution and stirring evenly to enhance the stability of the adjuvant; Filtering the mixed solution; The mixed solution was converted into a solid form by freeze drying.

4. The method for preparing a MPL composite adjuvant according to claim 3, characterized in that: The dissolution time of the MPL solution is 30-60 min, the dissolution temperature is 25-30° C., and the stirring rate is 300-600 rpm.

5. The method for preparing a MPL composite adjuvant according to claim 3, characterized in that: The dissolution time of the immunomodulatory peptide is 20-30 minutes, the dissolution temperature is 25-30° C., and the stirring rate is 200-300 rpm.

6. The method for preparing a MPL composite adjuvant according to claim 3, characterized in that: The preparation method of the nanocarrier comprises: The lipid is dissolved in a solvent, and the solvent is removed by rotary evaporation to obtain a nanocarrier, and the nanocarrier is subjected to surface functionalization treatment. The particle size of the nanocarrier is in the range of 50-200 nm.

7. The method for preparing a MPL composite adjuvant according to claim 6, characterized in that: The surface functionalization treatment of the nanocarrier comprises the following steps: The nanocarrier is treated with a PEGylation reagent, wherein the mass of the PEGylation reagent accounts for 0.1%-1.0% of the mass of the nanocarrier, the reaction temperature is 30-40° C., and the reaction time is 2-4 hours.

8. The method for preparing a MPL composite adjuvant according to claim 3, characterized in that: The filtering process comprises: Use a 0.22 μm filter membrane for pre-filtration to remove larger impurities and ensure the initial purity of the solution; Then, ultrafiltration with a pore size range of 0.05-0.2 μm is used to further remove tiny particles and microorganisms to ensure the purity and sterility of the composite adjuvant.

9. The method for preparing a MPL composite adjuvant according to claim 3, characterized in that: The composite adjuvant solution is frozen at -80°C for 1.5-2 hours, and then dried using a freeze-drying device for 10-12 hours.

10. Use of the MPL composite adjuvant according to claim 1 or 2 in vaccine development.

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