CN118340878A - Lovastatin-PLGA modified material based on PEI modification, preparation method thereof, pickering emulsion and application - Google Patents
Lovastatin-PLGA modified material based on PEI modification, preparation method thereof, pickering emulsion and application Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
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- Medicinal Preparation (AREA)
Abstract
The invention discloses a lovastatin-PLGA modified material based on PEI modification, a preparation method thereof, pickering emulsion and application, wherein the preparation method comprises the following steps: s100, mixing lovastatin and PLGA with an organic solvent to obtain an organic phase; s200, mixing the organic phase in the step S100 with water, performing ultrasonic treatment, and adding an emulsifying agent into the mixture for emulsification to prepare emulsion; s300, adding PEI into the emulsion in the step S200 under the stirring condition to prepare modified emulsion; s400, removing impurities from the modified emulsion to obtain the lovastatin-PLGA modified material based on PEI modification. The stable, safe and efficient Pickering emulsion constructed by adopting the emulsion granulation strategy can realize the co-loading and the cooperative delivery of the lovastatin and the antigen, and solve the problem of poor water solubility of the lovastatin.
Description
Technical Field
The invention relates to the field of vaccine adjuvant materials, in particular to a lovastatin-PLGA modified material based on PEI modification, a preparation method thereof, a Pickering emulsion and application thereof.
Background
Immunoprophylaxis with vaccines is one of the most effective means of intervention and control of infectious diseases in various animals. And adjuvants play a very critical role in the process of the vaccine exerting an effective immune effect.
There are a number of existing adjuvants, each having their own advantages and disadvantages, for example: the commonly used oil adjuvant has better adjuvant activity, but has the defects of strong toxic and side effects, easy formation of induration, granuloma and the like in local injection; the aluminum adjuvant can generate strong humoral immunity, but has poor effect on novel subunit vaccine, can not induce effective cellular immunity, and has poor effect on preventing and treating intracellular infection; the ISA series adjuvant has excellent effect on improving animal humoral and cellular immunity, but the cost is far higher than other adjuvants. Thus, there remains a need for further development of high activity, safe and low cost veterinary adjuvants.
With increasing importance of food safety, the search for effective immunoadjuvant from natural medicinal animals and plants is one of the trends of future adjuvant development. The existing adjuvants also include products developed from natural medicinal animals and plants, such as propolis adjuvant, and immunostimulating complex composed of Quila or Q21[6] extracted from Quila saponaria. The traditional Chinese medicine and the active ingredients thereof have long history in improving the immunity of the organism, and researches show that the main ingredients of a plurality of traditional Chinese medicines, such as saponin, polysaccharide and flavonoid ingredients, have good effect of improving the immunity of the organism. Modern researches show that the red rice can improve the nonspecific immune response level of organisms, wherein lovastatin is used as a main active ingredient of the traditional Chinese medicine red rice, has excellent adjuvant activity, but the clinical application of the lovastatin is limited because the lovastatin is difficult to dissolve in water.
Disclosure of Invention
Aiming at the prior art, the invention aims to solve the problems of poor water solubility, low bioavailability and strong side effect of the monascus lovastatin in the prior art, more defects in the application to vaccine adjuvants and the like, thereby providing a lovastatin-PLGA modified material based on PEI modification, a preparation method thereof and a pickering emulsion with high-efficiency and safe application effect.
In order to achieve the above object, the present invention provides a preparation method of lovastatin-PLGA modified material based on PEI modification, which comprises the following steps:
S100, mixing lovastatin and PLGA with an organic solvent to obtain an organic phase;
S200, mixing the organic phase in the step S100 with water, performing ultrasonic treatment, and adding an emulsifying agent into the mixture for emulsification to prepare emulsion;
S300, adding PEI into the emulsion in the step S200 under the stirring condition to prepare modified emulsion;
s400, removing impurities from the modified emulsion to obtain the lovastatin-PLGA modified material based on PEI modification.
Preferably, in step S100, the weight ratio of the lovastatin to the PLGA is 1:8-12;
and/or the organic solvent is selected from acetone.
Preferably, in step S200, the volume ratio of the organic phase to the water is 1:0.1-0.4;
and/or in step S200, the ultrasonic power of ultrasonic treatment in the process of mixing the organic phase and water is 5-30%, and the ultrasonic treatment time is 0.5-3min.
Preferably, in step S200, the emulsifier employed is block polyether F-68 (i.e. poloxamer 188);
And/or ultrasonic mixing is adopted in the emulsification process of adding the emulsifying agent.
Preferably, the ultrasonic power in the ultrasonic mixing is 5-30%, and the ultrasonic mixing time is 1-3min.
Preferably, the emulsifier is provided by an aqueous solution of the block polyether F-68, and the concentration of the aqueous solution of the block polyether F-68 is 0.5 to 1% by weight.
Preferably, the volume ratio of the organic solvent in step S100 to the aqueous solution of the block polyether F-68 in step S200 is 1:8-12.
Preferably, the weight ratio of the PLGA to the PEI is 8-12:1.
Preferably, the PEI is provided by an aqueous solution of PEI and the concentration of the aqueous solution of PEI is 80-120mg/mL.
Preferably, the impurity removal process in step S400 includes at least centrifugation, washing and freeze-drying processes sequentially performed.
The invention also provides a lovastatin-PLGA modified material based on PEI modification, which is prepared by adopting the preparation method.
The invention also provides a pickering emulsion, which contains the lovastatin-PLGA modified material based on PEI modification.
Preferably, the pickering emulsion is obtained by emulsifying the lovastatin-PLGA modified material based on PEI modification with an oil phase.
Preferably, the oil phase is selected from squalene.
In order to achieve the emulsification process, the PEI-based modified lovastatin-PLGA modified material herein is present in the form of an aqueous solution thereof. Specifically, the relatively pure lovastatin-PLGA modified material based on PEI modification prepared through the operation processes of centrifugation, washing, freeze-drying and the like needs to be further dissolved in water, and then an aqueous solution based on the lovastatin-PLGA modified material modified by PEI is obtained and is used as an aqueous phase and is further emulsified with the oil phase. Meanwhile, the mixing proportion of the lovastatin-PLGA modified material modified by PEI and water can be adjusted according to actual conditions, and the existence of water is mainly used for providing a medium for the existence of water phase so as to ensure the effective implementation of the subsequent emulsification process. For example, the amount of water is 7mL, and the amount of the corresponding PEI-modified lovastatin-PLGA modified material (in the form of a drug solution) before the removal of impurities (i.e., before the centrifugation, washing and lyophilization processes) is 15-25mL (it should be further explained that the aforementioned use of the drug solution before the removal of impurities as a metering standard for the amount is for the convenience of calculation, and when it is mixed with water as an aqueous phase, the corresponding lyophilized powder after the removal of impurities is actually used). It should be noted that the present invention is not limited to this specific range, and those skilled in the art can make corresponding adjustments according to actual situations, and will not be described herein.
Preferably, the ratio of the oil phase to the water phase is 9:1 to 5:5.
The invention also provides application of the Pickering emulsion as a vaccine adjuvant.
Aiming at the current situation that the prior monascus lovastatin has poor water solubility, low bioavailability and strong side effect and needs to be solved, the invention further adopts PEI for modification on the basis of emulsion prepared by mixing lovastatin and PLGA, and then the modified material is further subjected to impurity removal to prepare the pickering emulsion. Based on the method, the stable, safe and efficient Pickering emulsion constructed by adopting the emulsion granulation strategy can realize the co-loading and the cooperative delivery of the lovastatin and the antigen, and solve the problem of poor water solubility of the lovastatin. Meanwhile, the safety and high efficiency characteristics of the obtained pickering emulsion further promote the application of the monascus lovastatin adjuvant when the pickering emulsion is applied to the vaccine adjuvant.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 is a graph showing the results of the detection of the Pickering emulsion prepared in detection example 1 according to the present invention;
FIG. 2 is a graph showing the results of the stability test for Lov-PPE/OVA in test example 2 of the present invention; wherein, fig. 2A corresponds to the particle size variation trend, fig. 2B corresponds to the polydispersity index variation trend, and fig. 2C corresponds to the potential (zeta potential) variation trend;
FIG. 3 is a graph showing the results of antigen release from Lov-PPE/OVA in test example 3 of the invention;
FIG. 4 is a flow chart of mouse immunization and a graph of antibody level results after immunization according to verification example 1 of the present invention; wherein, fig. 4A is a flow chart of mouse immunization, fig. 4B is an IgG (B) antibody level after immunization, fig. 4C is an IgG1 (C) antibody level after immunization, and fig. 4D is an IgG2a (D) antibody level after immunization;
FIG. 5 is a graph showing the results of the activation of spleen lymphocytes in verification example 2 of the present invention;
FIG. 6 is a graph showing the results of the present invention for the verification of splenic cytotoxic T lymphocytes in example 2;
FIGS. 7A to 7D are quantitative cell analysis charts in verification example 3 of the present invention; wherein, FIG. 7A is a quantitative analysis and a representative flow cytometry graph of IFN gamma +、TNFα+ and IL4 +CD4+ T cells, FIG. 7B is a quantitative analysis and a representative flow cytometry graph of IFN gamma + and TNF alpha +CD8+ T cells, and FIG. 7C and FIG. 7D are cytokines produced by splenocytes after 2D re-stimulation with OVA;
FIGS. 8A to 8C are diagrams showing the results of security evaluation in verification example 4 of the present invention; wherein, fig. 8A is a histopathological change detection result diagram, and fig. 8B is a blood biochemical detection result diagram; FIG. 8C is a graph showing the results of the distribution detection of various immune cell subtypes in blood.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The technical scheme of the invention is described in detail below with reference to specific embodiments. Wherein, the lovastatin is the conventional commercial product with the product number of 75330-75-5 produced by Shanghai Mieli biological company, PLGA is the conventional commercial product with the product number of 14219-546 produced by Jinan Daida biological company, F68 (namely block polyether F-68) is the conventional commercial product with the product number of 9003-11-6 produced by Source leaf biological company, PEI is the conventional commercial product with the product number of 9002-98-6 produced by Sigma Aldrich biological company, and squalene is the conventional commercial product with the product number of 111-02-4 produced by Shanghai Mieli biological company.
Example 1 preparation of lovastatin-PLGA modified material based on PEI modification:
first, 5mg of lovastatin (Lov) and 50mg of PLGA were dissolved in 1mL of acetone. After mixing well, 200. Mu.L of ultrapure water was added, and the mixture was sonicated at 10% power for 1 minute. Subsequently, 10 ml of 0.7% F68 was added and the mixture was sonicated at 20% power for 2min to form a composite emulsion. After stirring the composite emulsion for 1h in magnetic suspension, 100mg/mL of aqueous PEI solution (the ratio of addition is such that PLGA: pei=10:1) was added in proportion, and magnetic stirring was continued for 3h to remove the organic solvent in the emulsion. The emulsion was centrifuged (12000 r/min,30 min), washed with ultrapure water and lyophilized to give a lovastatin-PLGA modified material based on PEI modification (denoted as Lov-PP, in the form of nanoparticles) which was stored at 4 ℃.
Example 2 preparation of pickering emulsion:
After lyophilization, the original 20mL of the drug solution (the drug solution described herein, i.e., the emulsion before centrifugation, ultra-pure water washing and lyophilization treatment in example 1; and in the case of use in this example 2, the lyophilized nanoparticles corresponding to the 20mL of the drug solution before treatment were dissolved in 7mL of deionized water as a practical use form), and squalene (as an oil phase) was emulsified with the Lov-PP nanoparticle suspension prepared in example 1 by ultrasonic waves (the ratio of addition was such that the oil-water ratio was 3:7) to obtain a pickering emulsion (Lov-PPE).
Application example
The pickering emulsion (Lov-PPE) prepared in example 2 was combined with OVA (ovalbumin as model antigen) in a volume ratio of 1:1, mixing at low speed for 5min by a vortex instrument, then placing in a constant temperature oscillator, mixing for 2h, enabling OVA to be loaded on Pickering emulsion to obtain Lov-PPE/OVA, and placing the prepared vaccine preparation at 4 ℃ for preservation.
Detection example 1
Particle size, polydispersity index (PDI) and Zeta potential of the pickering emulsion obtained in example 2 were measured using a Nano Zeta Sizer. The pickering emulsion was then diluted and examined for morphology using a freeze scanning electron microscope. Subsequently, lov-PP nanoparticles were labeled with FITC (green), squalene was stained with CY5 (pink) for identification by Confocal Laser Scanning Microscopy (CLSM). The results obtained are shown in FIG. 1. As shown in FIG. 1A, the average particle diameter is 1062+ -97 nm; as shown in fig. 1B, PDI is 0.13±0.149; as shown in fig. 1C, the Zeta potential is positive. The photomicrograph is shown in fig. 1D, and the low-temperature scanning electron microscope (cryo-SEM) image is shown in fig. 1E, wherein it can be seen from fig. 1E that Lov-PP is uniformly and densely distributed on the surface of pickering emulsion to form a stable emulsion, and the uniform distribution enhances the stability of the emulsion by forming a compact particle layer around the droplets. As shown in FIGS. 1F-1H, fluorescence confocal microscopy using Cy5 dye for squalene (pink) and FITC dye for LovPP/OVA (green) showed confocal phenomenon, indicating successful preparation of structurally stable, effective drug-loaded Lov-PPE/OVA.
Detection example 2
The Lov-PPE/OVA emulsion prepared in example 2 was stored in an environment of 4 ℃ and monitored for particle size, polydispersity index (PDI) and zeta potential weekly over a period of 5 weeks, and the results obtained are shown in fig. 2, wherein fig. 2A corresponds to the trend of particle size change, fig. 2B corresponds to the trend of polydispersity index change, and fig. 2C corresponds to the trend of potential (zeta potential) change. As can be seen from FIG. 2, after 5 weeks of storage at 4℃the particle size and zeta potential of the Lov-PPE/OVA did not change significantly, and the PDI remained below 0.3 all the time, which proves that the pickering emulsion was very stable throughout the storage period.
Detection example 3
The dialysis bag containing Lov-PPE/OVA nanoparticles was immersed in a beaker and rinsed with deionized water for 12h to eliminate unbound OVA antigen. Subsequently, the sample was treated with 2M sodium chloride at 1:1, and standing for more than 24 hours. Then, it was centrifuged at 12000rpm for 30min to separate the aqueous phase from the oil phase. The BCA protein assay kit was used to quantify the concentration of OVA antigen in the aqueous phase. The results obtained are shown in FIG. 3. As can be seen from FIG. 3, lov-PPE had a better controlled release effect, with over 75% of OVA released by day 21. This controlled release mechanism is very advantageous for vaccine applications because it ensures prolonged antigen availability, thereby helping to enhance and maintain immune responses.
Female Balb/c mice were placed in a sterile environment for one week after adaptation, and were divided into 6 groups, and each group was inoculated with PBS (i.e., phosphate buffer commonly used in the art as a control group), free OVA, alum/OVA (Alum specifically as aluminum adjuvant-bound OVA), lov/OVA (i.e., lovastatin-bound OVA), PPE/OVA (i.e., lovastatin-free blank nanoparticle-bound OVA) and Lov-PPE/OVA (i.e., lov-PPE-bound OVA prepared in the application example), followed by booster immunization at 14d at 200. Mu.L each immunization dose.
Specifically, lov-PPE/OVA: mixing the prepared Lov-PPE and OVA antigen according to a volume ratio of 1:1 for 5min, then mixed in a constant temperature shaker for 2h, and stored at 4 ℃ (Lov, 1mg/mL, OVA, 500. Mu.g/mL).
PPE/OVA: the prepared PPE and OVA antigen are mixed according to the volume ratio of 1: 1. Mixing at low speed for 5min with vortex, mixing in a constant temperature shaker for 2h, and storing the prepared vaccine preparation at 4deg.C (OVA, 500 μg/mL).
Lov/OVA: after mixing Lov solution with OVA antigen by vortexing for 5min, shaking and mixing for 2h, the prepared vaccine preparation was kept at 4deg.C (Lov, 1mg/mL, OVA, 500. Mu.g/mL).
Alum/OVA: aluminum adjuvant and OVA antigen are mixed according to the volume ratio of 1: 1. Vortex mixing for 5min, then shake mixing for 2h, and store the prepared vaccine formulation at 4deg.C (Alum, 20mg/mL, OVA,500 μg/mL).
OVA: the OVA antigen solution and PBS solution are mixed according to the volume ratio of 1: 1. Vortex mixing for 5min, then shake mix for 2h, and place the prepared vaccine formulation at 4deg.C for storage (OVA, 500 μg/mL).
Control: PBS solution was used as control.
Further, at 28d after immunization, 4 mice were randomly selected from each group, spleens of the selected mice were collected, ground, centrifuged, red blood cells were lysed with a red blood cell lysate and washed, and spleen lymphocytes were isolated to prepare a spleen cell suspension. Each sample of these suspensions contained 5X 10 5 cells, cultured in RPMI-1640 medium containing 10% fetal bovine serum and 3% penicillin-streptomycin. Cultures were re-stimulated with 50. Mu.g/mL OVA antigen and incubated at 37℃for 48h.
And further carrying out the following verification example on the basis:
verification example 1, determination of IgG antibodies and different subclasses of antibodies:
Serum samples were taken from each group of mice at 21d, 28d, 35d, 42d and 49d after the primary immunization, and levels of OVA-specific IgG antibodies and IgG1, igG2a subclasses antibodies were detected by means of enzyme-linked immunosorbent assay (ELISA). The results obtained are shown in FIG. 4. As can be seen from FIG. 4, the levels of IgG and its subclass antibodies were significantly increased in the serum of mice immunized with Lov-PPE as an adjuvant, compared to the other groups. Wherein, fig. 4A is a flow chart of mouse immunization, fig. 4B is an IgG (B) antibody level after immunization, fig. 4C is an IgG1 (C) antibody level after immunization, and fig. 4D is an IgG2a (D) antibody level after immunization.
Verification example 2, activation of splenic lymphocytes and determination of cytotoxic T cells:
Spleen lymphocytes (i.e., cultures re-stimulated and incubated with OVA antigen) after 48h of antigen re-stimulation were stained with FITC-CD3, APC-CD4, PE-CD8, and PE-Cyanine7-CD69 antibodies and assayed for activation status by flow cytometry. The results obtained are shown in FIG. 5.
Spleen lymphocytes after 48 hours of antigen re-stimulation were stained with PE-Cyanine7-CD3, PE-CD8, FITC-CD107a and APC-CD178 antibodies and analyzed by flow cytometry to determine the proportion of cytotoxic T lymphocytes in the spleen. The results obtained are shown in FIG. 6.
From fig. 5, it can be seen that the activation level of antigen-specific T cells in the spleen of mice, wherein fig. 5A is a graph of flow results corresponding to fig. 5A for detecting the proportion of CD3 +、CD4+、CD69+ cells in the 28D spleen sample after primary immunization by flow cytometry, fig. 5C is a graph of flow results corresponding to fig. 5A, fig. 5B is a graph of flow results corresponding to fig. 5C for detecting the proportion of CD3 +、CD8+、CD69+ cells in the 28D spleen sample after primary immunization by flow cytometry. The ratio of Lov-PPE group CD4 + T cell activation (CD 3 +、CD4+、CD69+) to CD8 + T cell activation (CD 3 +、CD8+、CD69+) was 13.73% and 11.41%, respectively, significantly higher than the other groups. This suggests that Lov-PPE significantly enhances activation of CD4 + and CD8 + T lymphocytes, thereby enhancing immune responses.
Further, as shown in FIG. 6 (wherein FIG. 6A is a graph of the results of flow cytometry for detecting the proportion of CD3 +、CD8+、CD107a+ cells in the spleen sample at 28D after primary immunization; FIG. 6C is a graph of the results of flow cytometry corresponding to FIG. 6A, FIG. 6B is a graph of the results of flow cytometry for detecting the proportion of CD3 +、CD8+、CD178+ cells in the spleen sample at 28D after primary immunization; FIG. 6D is a graph of the results of flow cytometry corresponding to FIG. 6B). The groups treated with Lov and Lov-PPE showed significant up-regulation of CD107a and CD178 on CD8 + T cells (p < 0.05) compared to OVA groups. This suggests that CTLs are strongly activated, highlighting the efficacy of Lov and Lov-PPE as adjuvants in promoting their cytotoxic function on pathogen-infected cells. In contrast, the group receiving alum adjuvant did not significantly increase CTL activation compared to OVA alone. While aluminum adjuvants widely used in vaccine formulations may not be as effective at eliciting a strong CTL response, further highlighting the potential of Lov-PPE as a vaccine adjuvant.
Verification example 3, determination of intracellular cytokines and determination of cell supernatant cytokines:
After 48h of restimulation of spleen lymphocytes, cells were stained with FITC-CD3, APC-CY7-CD4 and BV605-CD 8. Cells were then fixed, broken, and stained with PE-TNFα, BV421-IFNγ, and PerCP-Cy5.5-IL4 antibodies, and analyzed by flow cytometry to assess the ability of spleen lymphocytes to secrete cytokines. To further examine the cytokine produced after re-stimulation of spleen lymphocytes, we cultured prepared spleen lymphocytes in vitro and re-stimulated with OVA (50. Mu.g/ml) for 48h, and the cell supernatants were collected and assayed for cytokine content by ELISA. The results are shown in FIG. 7, wherein FIG. 7A is a flow cytometry graph of quantitative analysis and representation of IFN gamma +、TNFα+ and IL4 +CD4+ T cells, FIG. 7B is a flow cytometry graph of quantitative analysis and representation of IFN gamma + and TNF alpha +CD8+ T cells, and FIG. 7C and FIG. 7D are cytokines produced by splenocytes after 2D re-stimulation with OVA. Quantitative cytokines (unit: pg/mL) included Th1 cytokines (IFNγ, TNFα, corresponding to FIG. 7C) and Th2 cytokines (IL 4, IL6, corresponding to FIG. 7D).
As shown in FIG. 7, lov-PPE vaccinated mice showed a higher frequency of cytokine-producing CD4 + T cells compared to the control group (FIG. 7A). Similarly, we also observed a significant increase in cytokine-producing CD8 + T cells following restimulation (fig. 7B). To further determine the immune response elicited by the Lov-PPE platform, we quantified cytokine secretion from spleen cells after two days of restimulation with OVA (fig. 7C). Th1 cytokines (including IFNγ and TNFα) were secreted by Lov-PPE treated mice at significantly higher levels than most controls. In addition, th2 cytokine (e.g., IL4 and IL 6) levels were also elevated in mice vaccinated with Lov-PPE compared to the control group. Lov-PPE can induce unique cytokines such as IFNgamma, TNF alpha, IL4, IL6 and the like, and shows the capability of stimulating Th1/Th2 comprehensive immune response.
Verification example 4, safety evaluation after immunization:
the security evaluation result is shown in fig. 8. Specifically:
Mice were euthanized 49d after primary immunization, hearts, livers, spleens, lungs and kidneys of the mice were collected, sectioned and stained (hematoxylin and eosin staining followed by fixation with 4% paraformaldehyde) to detect histopathological changes, the results of which are shown in fig. 8A, and it was seen that no apparent pathological changes occurred in the examined organ, confirming that the integrity and functionality of the organ structure were preserved. This suggests that Lov-PPE does not adversely affect critical organs, thus demonstrating its safety.
To further evaluate the effect of Lov-PPE on the whole body, blood biochemical analysis was performed. Specifically, blood was collected from mice 49d after the primary immunization, and blood biochemical tests were performed to examine whether or not aspartic acid Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP) and Lactate Dehydrogenase (LDH) were abnormal in each group of mice, and the results obtained are shown in fig. 8B. As can be seen from FIG. 8B, the levels of these enzymes remained within normal physiological ranges, indicating that administration of Lov-PPE did not cause hepatotoxicity or nephrotoxicity.
The results of examining whether the distribution of various immune cell subtypes (leukocyte, monocyte, lymphocyte, basophil, eosinophil, neutrophil and other related immune cells) in blood after the initial immunization of 49d of the mouse was abnormal are shown in fig. 8C. As can be seen from FIG. 8C, there was no significant difference between these cell populations in Lov-PPE/OVA immunized mice and the control group. This result suggests that Lov-PPE does not disrupt the distribution or function of immune cells, which is a critical consideration for the safety assessment of any vaccine adjuvant. In summary, extensive safety assessment of Lov-PPE (including histopathological, biochemical and hematological analysis) ultimately demonstrated its safety and biocompatibility as an in vivo adjuvant, highlighting the potential of Lov-PPE as a safe and effective vaccine formulation ingredient.
According to the technical scheme, the pickering emulsion prepared based on the PEI modified lovastatin-PLGA modified material can be used as an immunoadjuvant. Specifically, the serum antibody level of the OVA immunized mice can be obviously improved, the proportion of functional CD4 + T cells, the proportion of functional cytokine level and multifunctional cytokine secretion T cells can be obviously improved, th1 reaction can be promoted, th2 reaction can be promoted, and thus, the hybrid immunoprotection capability is generated. Lov-PPE can also promote the activation of T cells, induce the differentiation of killer T cells and effectively enhance the killing effect of CTL. In addition, the mice did not show systemic toxicity after immunization, indicating that it is a safe and efficient vaccine adjuvant.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. The preparation method of the lovastatin-PLGA modified material based on PEI modification is characterized by comprising the following steps of:
S100, mixing lovastatin and PLGA with an organic solvent to obtain an organic phase;
S200, mixing the organic phase in the step S100 with water, performing ultrasonic treatment, and adding an emulsifying agent into the mixture for emulsification to prepare emulsion;
S300, adding PEI into the emulsion in the step S200 under the stirring condition to prepare modified emulsion;
s400, removing impurities from the modified emulsion to obtain the lovastatin-PLGA modified material based on PEI modification.
2. The method of claim 1, wherein in step S100, the lovastatin and PLGA are used in a weight ratio of 1:8-12;
and/or the organic solvent is selected from acetone.
3. The method according to claim 1 or 2, wherein in step S200, the volume ratio of the organic phase to the water is 1:0.1-0.4;
and/or in step S200, the ultrasonic power of ultrasonic treatment in the process of mixing the organic phase and water is 5-30%, and the ultrasonic treatment time is 0.5-3min.
4. The method according to claim 1 or 2, wherein in step S200, the emulsifier used is block polyether F-68;
And/or ultrasonic mixing is adopted in the emulsification process of adding the emulsifying agent;
Preferably, the ultrasonic power in the ultrasonic mixing is 5-30%, and the ultrasonic mixing time is 1-3min.
5. The preparation method according to claim 4, wherein the emulsifier is provided by an aqueous solution of the block polyether F-68, and the concentration of the aqueous solution of the block polyether F-68 is 0.5 to 1 wt%;
preferably, the volume ratio of the organic solvent in step S100 to the amount of the aqueous solution of F-68 in step S200 is 1:8-12.
6. The method of claim 1 or 2, wherein the weight ratio of PLGA to PEI is 8-12:1;
preferably, the PEI is provided by an aqueous solution of PEI and the concentration of the aqueous solution of PEI is 80-120mg/mL.
7. The preparation method according to claim 1 or 2, wherein the impurity removal process in step S400 includes at least centrifugation, washing and freeze-drying processes sequentially performed.
8. Lovastatin-PLGA modified material based on PEI modification, characterized in that it is prepared by the preparation method according to any one of claims 1 to 7.
9. A pickering emulsion comprising the PEI-modified lovastatin-PLGA-based modified material as defined in claim 8;
Preferably, the pickering emulsion is obtained by emulsifying the lovastatin-PLGA modified material based on PEI modification with an oil phase;
Preferably, the oil phase is selected from squalene;
Preferably, the PEI-modified lovastatin-PLGA-based modified material involved in the emulsification process is present in the form of an aqueous solution thereof and is formed into an aqueous phase;
Preferably, the ratio of the oil phase to the water phase is from 9:1 to 5:5.
10. Use of the pickering emulsion of claim 9 as a vaccine adjuvant.
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| US20160175432A1 (en) * | 2014-06-18 | 2016-06-23 | Institute Of Process Engineering, Chinese Academy Of Sciences | An oil-in-water emulsion containing no surfactant and use thereof |
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