Patent: Preparation and application of pachyman nano adjuvant and adjuvant/antigen co-delivery vaccine based on graphene oxide, belonging to the field of medicines.
The invention comprises a pachyman nano adjuvant which is formed by taking a nano graphene oxide material as a carrier and pachyman loaded on the carrier, and an adjuvant/antigen co-delivery vaccine formed by the adjuvant and an antigen.
The pachyman nanometer adjuvant can promote dendritic cell maturation, enhance lymphocyte function, facilitate drug release, effectively prolong drug effect, prevent immune tolerance, and greatly enhance immune effect and reaction time.
The adjuvant/antigen co-delivery vaccine enhances the bioavailability of pachyman and antigen, enables the antigen and the adjuvant to be ingested by the same cell, greatly enhances the targeting property of the vaccine, and can induce not only humoral immunity but also stronger cellular immunity. The invention is used as a novel adjuvant and vaccine, and can be expected to be used for preventing and treating human diseases.
The invention belongs to the field of medicines, and particularly relates to a pachyman nano adjuvant based on a graphene oxide material, and a preparation method and application of an adjuvant/antigen co-delivery vaccine formed by the pachyman nano adjuvant and an antigen.
The development and application of vaccines has been expanded from disease prevention to the treatment of many non-infectious diseases, and traditional attenuated live vaccines generally have high immunogenicity, but risk of back mutation, and the immune effect has certain limitations. Inactivated vaccines, subunit vaccines, DNA vaccines and recombinant vaccines are relatively safe, but have limited immunogenicity, and can exert long-term effective protective effects by being supplemented with adjuvants.
The immunoadjuvant, as a nonspecific immunopotentiator, can significantly enhance the immunological effect or change the type of immune response after vaccination by being injected into the body in advance or simultaneously with an antigen. Since the aluminum adjuvant was approved in the 20 th century, it has become the most widely used adjuvant, and it can enhance primary immunity, reduce antigen dose and immunization frequency, but cannot induce CTL response, and may cause various adverse reactions.
With a slow progress of decades, oil-in-water emulsion MF59 was approved for marketing at the end of the 90 s of the 20 th century, and AS03, AS01, AS04 and CpG ODN adjuvants have been approved in sequence since the 21 st century. In addition to the few vaccine adjuvants mentioned above, which are approved for human use, most of them are still in clinical or preclinical trial stage, and the number of adjuvants available for human use is very limited, so that the development of novel adjuvants is urgently needed.
Tuckahoe (Poria cos (Schw.) Wolf) is a long-standing traditional Chinese medicine and is widely applied to the treatment of traditional Chinese medicines. Poria cocos (Schw.) wolf of Polyporaceae, Polyporus fungi, has dry sclerotium, which grows mainly on the root of pine tree. The active ingredients mainly comprise: polysaccharides, alkaloids, saponins, terpenes, polyphenols, etc.
The polysaccharides are the main components of Poria cocos, and the pachyman is a mixture of different types of polysaccharides extracted and purified from Poria cocos, and its content is about 84% of Poria cocos dry sclerotium, and it includes water-soluble polysaccharides and alkali-soluble polysaccharides, and is composed of glucose, fucose, arabinose, xylose, mannose and galactose.
A large number of researches show that pachyman has the effects of enhancing immunity, protecting immune organs of organisms, enhancing functions of T cells and B cells, influencing the activity of dendritic cells and influencing the release of certain cytokines, thereby promoting specific and non-specific immune response. And the low toxicity and fewer side effects of pachyman make it an ideal immunological adjuvant. However, the molecular weight of pachyman is large, and the drug release is too fast, which can cause overlarge injection dosage, thereby increasing the difficulty of clinical application of pachyman. Therefore, it is urgently required to solve this problem.
Nanomaterial refers to single crystals or polycrystalline bodies with grain size less than 100 nm. The nanoparticles have biological characteristics of being easily taken up by various cells, and can be phagocytized by antigen presenting cells, so that the immune response caused by antigens is enhanced. The nano material can also slowly release the antigen, reduce the dosage or times of the antigen, increase the size of the small molecule antigen, and fully process and treat the antigen, so that the immune effect is more durable.
Some nanoparticles themselves have a stimulatory effect on the immune system and immunization with vaccines containing nanomaterials can result in an immune response in the mucosal and gastrointestinal mucosa. Graphene is a carbon nano-material with a two-dimensional plane structure, and the special monoatomic layer structure of the graphene enables the graphene to have a plurality of unique physicochemical properties.
Graphene oxide is an oxide of graphene, and since graphene oxide contains more oxygen-containing functional groups after oxidation, properties of graphene oxide can be improved by various reactions with the oxygen-containing functional groups. The graphene oxide and the derivatives thereof have the advantages of high specific surface area, strong electric and thermal conductivity, good biocompatibility and the like, and are widely applied to the biomedical fields of drug carriers, cancer detection and treatment and the like.
Therefore, pachyman is needed to be selected as a raw material, a graphene oxide coating method is adopted, the medicine is slowly released, the dosage of the pachyman is reduced, and an effective, safe and stable immunopotentiator, namely the pachyman nano adjuvant based on the graphene oxide material, is found.
The invention provides a pachyman nano adjuvant and an adjuvant/antigen co-delivery vaccine formed by the adjuvant. The pachyman nanometer adjuvant can greatly enhance the immune effect of vaccines, reduce the dosage of pachyman, reduce the dosage of vaccine antigens after the antigens are encapsulated, and solve various limited problems in clinical application in the prior art.
The invention also provides a pachyman nano adjuvant and a preparation method of the adjuvant/antigen co-delivery vaccine formed by the adjuvant, and the preparation method is simple and easy to operate, has obvious effect and is suitable for large-scale production.
In order to achieve the purpose, the invention adopts the following technical scheme:
The invention provides a pachyman nano adjuvant, wherein a carrier material of the pachyman nano adjuvant is nano graphene oxide, and pachyman is connected to a nano graphene oxide carrier to form the pachyman nano adjuvant based on a graphene oxide material. The pachyman nanometer adjuvant provided by the invention can load pachyman with high efficiency, and solves the clinical application problems of quick release of pachyman medicine and overlarge injection dosage.
The nano graphene oxide is a graphene oxide treated by a nano method, has more oxygen-containing functional groups, and is a nano material with high specific surface area and good biocompatibility. The particle size is 1-100 nm, the molecular weight is 5-10 kDa, the carrier can enter lymph nodes to act, and the carrier is widely applied to the field of carriers of medicines and vaccines.
The molecular weight of the pachyman is 8-15 kDa, the constitutional monosaccharides of the pachyman comprise glucose, mannose, fucose, galactose and the like, and the mass ratio of the constitutional monosaccharides is (2-3.5): (1-2): (0.3-4): 1.
The mass ratio of the nano graphene oxide to the pachyman is 1: 2.5-1: 10, preferably 1: 5.
the preferred particle size of the pachyman nanometer adjuvant is 100-500 nm, and the molecular weight is 15-45 kDa.
A method for preparing pachyman nanometer adjuvant comprises the following steps:
(1) dissolving and dispersing graphene oxide by using sterile water, carrying out ultrasonic treatment for 2 hours, carrying out ice bath ultrasonic treatment for 30 minutes, adding sodium hydroxide (the final concentration is 5M), and continuing ultrasonic treatment for 2 hours; centrifuging at a high speed by 16,000g of supergravity, and collecting supernatant to obtain a nano graphene oxide carrier solution, wherein the final concentration of the nano graphene oxide carrier solution is 0.1-10 mg/mL, and preferably 1 mg/mL;
(2) and (3) carrying out ultrasonic treatment on the nano graphene oxide solution in the step (1) for 1 hour, and adjusting the pH to 9-10, preferably 9.5. Adding epoxy chloropropane, introducing nitrogen for reaction, stirring and reacting for 4 hours at the temperature of 40 ℃ in water bath, and dialyzing to remove unreacted epoxy chloropropane;
(3) preparing pachymaran solution with deionized water, and performing endotoxin-removing affinity chromatography treatment, wherein the endotoxin content is less than 5EU/mL, and the concentration of the pachymaran solution is 2-20 mg/mL, preferably 5 mg/mL.
(4) And (3) dissolving the pachymaran solution in the nano graphene oxide solution in the step (2), adjusting the pH to 9-10, preferably 9, carrying out water bath at 42 ℃ for 1-3 hours, preferably 3 hours, and centrifuging and collecting to obtain the pachymaran nano adjuvant carrying the nano graphene oxide.
The pachymaran adjuvant prepared by any one of the preparation methods is also within the protection scope of the invention.
The invention also provides an adjuvant/antigen co-delivery vaccine, which is prepared by loading the pachyman nano adjuvant with a virus antigen. The antigen and pachyman of the adjuvant/antigen co-delivery vaccine are both connected with graphene oxide. The particle size of the vaccine is 50 nm-1000 nm, and the absolute value of zeta potential is 20-30 mv.
The viral antigens include: the virus antigen is any one of EV71 inactivated virus, hepatitis A inactivated virus, polio inactivated virus, influenza inactivated virus, HPV virus-like particles, hepatitis B virus-like particles or recombinant proteins containing the virus antigens.
The mass ratio of the nano graphene oxide to the pachyman is 1: 2.5-1: 10, and preferably 1: 5.
The mass ratio of the virus antigen to the pachyman is 1: 2-1: 50, and preferably 1: 25.
In the invention, the preparation method of the adjuvant/antigen co-delivery vaccine comprises the following steps:
(1) dissolving graphene oxide in sterile water, carrying out ultrasonic treatment for 2 hours, carrying out ice bath ultrasonic treatment for 30 minutes, adding strong base, carrying out continuous ultrasonic treatment for 2 hours, carrying out high-speed centrifugation at a hypergravity of 16,000g, and collecting supernatant to obtain a nano graphene oxide carrier solution, wherein the final concentration of the nano graphene oxide carrier solution is 0.1-10 mg/mL. Preferably 1 mg/mL.
(2) Carrying out ultrasonic treatment on the nano graphene oxide solution in the step (1) for 1 hour, adjusting the pH value to 9-10, preferably 9.5, adding epoxy chloropropane, introducing nitrogen for reaction, stirring and reacting at the temperature of 40 ℃ in a water bath for 4 hours, and dialyzing to remove unreacted epoxy chloropropane;
(3) preparing pachyman solution with deionized water, and performing endotoxin removal treatment, wherein the concentration of the pachyman solution is 2-20 mg/mL, and preferably 5 mg/mL.
(4) And (3) dissolving the pachymaran solution in the step (3) into the nano graphene oxide solution in the step (2), adjusting the pH to 9-10, preferably 9, carrying out water bath at 42 ℃ for 1-3 hours, preferably 3 hours, and centrifuging and collecting to obtain the pachymaran nano adjuvant coated with nano graphene oxide.
(5) Adjusting the pH of the pachyman nano adjuvant solution carrying nano graphene oxide in the step (4) to 4.5-7.2, preferably 6; adding EDC and Sulfo-NHS, and mixing for 15 min-1 h, preferably 30 min; adjusting the pH value to 7-8, preferably 7.2, adding a virus antigen solution, mixing for 1-5 h, preferably 2h, desalting by a desalting column to obtain an adjuvant/antigen co-delivery vaccine;
in the step (4), the EDC/Sulfo-NHS molar ratio is 1: 2-10: 1, preferably 4: 1. The EDC/graphene oxide concentration ratio is 1: 5-1: 20, preferably 1: 10.
in the step (4), the concentration of the virus antigen solution is 0.1-20 mg/mL, preferably 1 mg/mL.
The use of the antigen/adjuvant co-delivery vaccine described above in the preparation of a medicament is also within the scope of the present invention. For use in the field of prophylactic and therapeutic vaccines.
The raw materials of the kit used by the invention are commercially available; for the devices, conditions (temperature, time, etc.), substances, amounts, methods, etc., which are not specifically described in the present invention, any of those known in the art or those ordinarily skilled in the art can be used to determine the same.
Compared with the prior art, the invention has the following advantages:
1. The preparation of the pachyman nano adjuvant is not reported at home and abroad, and the invention provides a preparation method of the pachyman nano adjuvant based on a graphene oxide material and optimized conditions thereof.
2. The immunopotentiation activity of the pachymaran nanometer adjuvant is not reported. The implementation of the invention proves that the immune effect is greatly enhanced after the pachyman is prepared into the pachyman nanometer adjuvant, which is expressed by stimulating the dendritic cells of mice to mature in vitro and inducing TH1 type immune response. Therefore, the pachyman nano adjuvant provides materials and demonstration for the development of novel adjuvants.
3. The adjuvant/antigen co-delivery vaccine containing the pachyman nano adjuvant provided by the invention has high stability, greatly enhances the bioavailability of pachyman and antigen, enables the antigen and the adjuvant to be taken by the same cell, enhances the targeting property of the vaccine, can enhance TH1 type immune response, and has the advantages of strong immune response persistence, good immune effect, increased cytokine secretion and the like.
Figure 1: shows the preparation process of pachyman nano adjuvant and adjuvant antigen co-delivery vaccine.
Figure 2: shows Zeta potential of pachyman nano adjuvant.
FIG. 3: shows electron microscopy of pachyman nanoadjuvant.
Figure 4: shows that the pachyman nano-adjuvant vaccine was taken up by dendritic cells.
Figure 5: shows that pachyman nano-adjuvant vaccine induced BMDC up-regulated CD86 expression.
Figure 6: shows that pachyman nano-adjuvant vaccine induced BMDC up-regulated CD80 expression.
Figure 7: shows that pachyman nano-adjuvant vaccine induced BMDC to up-regulate MHCII expression.
Figure 8: shows that pachyman nano-adjuvant vaccine induced antigen-specific IgG antibody production after immunization of mice.
FIG. 9: shows that the Pachymaran nanometer adjuvant vaccine induces spleen cells to secrete IFN-gamma cytokines after mice are immunized.
FIG. 10: shows that the IL-4 cytokine secretion by splenocytes was induced after immunization of mice with the Pachymaran nanometer adjuvant vaccine.
The present invention is further illustrated by the following examples, which include, but are not limited to, the following examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The embodiment is a preparation process of the pachyman nano adjuvant based on the graphene oxide material and the adjuvant/antigen co-delivery vaccine formed by the adjuvant.
The preparation method comprises the following steps:
(1) dissolving graphene oxide in sterile water, carrying out ultrasonic treatment for 2 hours, carrying out ice bath ultrasonic treatment for 30 minutes, adding strong base, and carrying out ultrasonic treatment for 2 hours. Centrifuging at a high speed of 16,000g, and collecting supernatant to obtain a nano graphene oxide carrier solution with a final concentration of 1 mg/mL.
(2) And (2) carrying out ultrasonic treatment on the nano graphene oxide solution in the step (1) for 1 hour, adjusting the pH value to 9.5, adding epoxy chloropropane, introducing nitrogen for reaction, stirring in a water bath at 40 ℃ for reaction for 4 hours, and dialyzing to remove the unreacted epoxy chloropropane.
(3) Preparing pachyman solution with deionized water, and removing endotoxin to obtain pachyman solution with concentration of 5 mg/mL.
(4) And (3) dissolving the pachymaran solution in the step (3) in the nano graphene oxide solution in the step (2), adjusting the pH value to 9, carrying out water bath at 42 ℃ for 3 hours, and carrying out centrifugal collection to obtain the pachymaran nano adjuvant coated with nano graphene oxide.
(5) And (4) adjusting the pH of the pachyman nanometer adjuvant solution carrying the nanometer graphene oxide in the step (4) to 6. Ligation EDC and Sulfo-NHS were added and mixed for 30 min. Adjusting the pH value to 7, adding a virus antigen solution, mixing for 2 hours, desalting by a desalting column to obtain an adjuvant/antigen co-delivery vaccine;
the method for preparing the blank graphene oxide carrier is the same as the above, except that pachyman and antigen are not added.
The adjuvant/antigen co-delivery vaccine construction scheme based on pachyman nano adjuvant is shown in figure 1,
example 2 characterization of adjuvant/antigen Co-delivered vaccines with Pachymaran Nano adjuvant
(1) Measurement of surface Zeta potential
Pachymaran nano adjuvant/antigen co-delivered vaccine sample solutions were prepared precisely at a concentration of 0.1mg/mL using Milli-Q ultrapure water. 1mL of the sample solution was used to determine the surface Zeta potential of the nanoparticles using a Zeta potential analyzer (Malvern, UK, Zetasizer NanoZS). The temperature is kept for 20min before each detection, each sample is tested for three times, the result is averaged, the result is shown in figure 2, and the zeta potential absolute value is more than 20Mv, which indicates that the sample is relatively stable.
(2) Observation by transmission electron microscope
A pachyman nano adjuvant/antigen co-delivered vaccine sample solution with the concentration of 0.1mg/mL is precisely prepared by Milli-Q ultrapure water, 10-20 mu L of the sample solution is dripped on a 230-mesh copper net containing a carbon support film, and after drying at a constant temperature, the morphology of the sample is observed by a transmission electron microscope (TEM, FEI in USA, Tecnai G220S-TWIN, 200kV) (figure 3).
(3) Adjuvant/antigen Co-delivery vaccine stability Studies
The Zeta potential of an adjuvant/antigen co-delivered vaccine containing a proper amount of pachyman nano adjuvant is measured by a laser particle sizer, the same batch of adjuvant/antigen co-delivered vaccine is taken after two weeks, the Zeta potential is measured by the same method, no obvious difference exists, and the adjuvant/antigen co-delivered vaccine disclosed by the invention is good in stability.
(4) In vitro drug delivery
Taking a proper amount of adjuvant/antigen co-delivery vaccine containing pachyman nano adjuvant, wherein a mode antigen OVA is marked by FITC and then is added into DC2.4 mouse dendritic cells, after culturing for 6h, the cells are fixed, DAPI is adopted to stain cell nuclei, fluorescence is observed under a fluorescence microscope, as shown in figure 4, green fluorescence expression is seen outside the cell nuclei, part of dendritic cells take up the antigen, and the adjuvant/antigen co-delivery system can carry the antigen into the cells.
(5) Drug loading measurement
Taking a proper amount of adjuvant/antigen co-delivery vaccine containing pachyman nano adjuvant, and measuring the load capacity of antigen protein by using a BCA method, wherein the load concentration of the antigen protein is 0.5mg/mL, and the drug load capacity is good.
(a) The adjuvant/antigen co-delivery vaccine prepared in example 1, pachyman, blank nano graphene oxide carrier prepared in example 1, model antigen and PBS solution group were used as negative control (PBS) to treat mouse bone marrow-derived dendritic cells (BMDC), and the effect of the negative control on mouse dendritic cell maturation in vitro was explored as follows:
SPF grade C57/BL6 mice, female, were selected for 6-8 weeks.
(1) Preparation of bone marrow cells
Killing a mouse by using a cervical dislocation method, shearing skin and hair of the mouse, taking thighbone, soaking in 70% alcohol for 2-5 min, transferring to an RPMI1640 full serum culture medium (on ice), carefully shearing two ends of the thighbone, sucking 100-200 mu L of RPMI1640 by using a syringe each time, and repeatedly flushing bone marrow to a sterile tube containing the RPMI1640 until the bone is completely whitened. The bone marrow wash was transferred from the tube to a petri dish containing RPMI1640 whole serum by filtration through a 200 mesh nylon mesh. Culturing in cell culture box for 30 min. Nonadherent or loosely adherent cell counts were collected, adjusted to 1X106 cells/mL, 20ng/mL GM-CSF was added and plated.
(2) Culture of bone marrow-derived dendritic cells
The next day of bone marrow cell culture, the fluid was partially changed and new RPMI1640 whole serum medium containing GM-CSF was added. On the third day, the solution was completely changed, the cell supernatant was aspirated off, the nonadherent cells were gently washed away, and a new RPMI1640 whole serum medium containing GM-CSF was added to each well. On the fourth or fifth day, nonadherent or loosely adherent cells were collected by washing and adherent cells were discarded. On day six or day seven, after 48h of culture, immature BMDCs were collected for subsequent testing.
(3) Flow cytometry for determination of cell surface CD80 and CD86, MHCII
Immature BMDCs were collected and seeded at a cell concentration of 1X 106/mL in 24-well plates. Respectively adding pachymaran, adjuvant/antigen co-delivery vaccine, graphene oxide carrier, model antigen and PBS, treating for 24h, collecting incompletely attached cells, sealing for 30min by using anti-mouse CD16/32 antibody, adding APC anti-mouse CD80, PE/Cy7 anti-mouse CD86 and PE anti-mouse MHC II antibody, and dyeing for 30min to 1h at 4 ℃ in dark place. And collecting cells, and detecting by a flow cytometer.
The experimental results are shown in fig. 5-7, the graphene oxide carrier has no significant influence on the expression of BMDC surface marker molecules, and pachyman can significantly up-regulate CD86 and MHCII molecules on the BMDC surface. Adjuvant/antigen co-delivered vaccines can effectively induce BMDC maturation in vitro.
(a) The adjuvant/antigen co-delivered vaccine prepared in example 1, pachyman, model antigen and PBS solution group were used as negative control (PBS for short) to immunize mice, and the influence of the negative control on mouse cellular immunity and humoral immunity in vivo was explored, as follows:
c57BL/6 mice (female, 6-8w) were immunized subcutaneously on day D0, and each group was vaccinated with PBS, model antigen (10 μ g), pachyman (500 μ g) or a co-delivered vaccine containing 10mg of model antigen. Blood is taken after 2 weeks for storage, the boosting immunization is carried out once, the mice are sacrificed after 4 weeks, and the blood is taken for detecting the specific IgG antigen of the serum anti-mode antigen OVA of the mice respectively.
Spleen is taken and ground by a 70 mu m cell screen and washed by PBS/EDTA to prepare the spleen cell single cell suspension. After counting the cells, adding the cells into a 96-well plate according to 2 × 105/well, adding 300 μ g/mL pattern antigen solution, continuing culturing for 72h, collecting cell supernatant, and detecting the content of IFN-gamma and IL-4 cytokines in the cell supernatant by ELISA.
As shown in figure 8, the adjuvant/antigen co-delivered vaccine significantly increased the expression of the pattern antigen-specific IgG antibodies in mouse serum compared to the control group, indicating that humoral immunity could be promoted.
The adjuvant/antigen co-delivered vaccine significantly increased IFN- γ secretion by mouse splenocytes compared to the control group, indicating that cellular immune responses could be enhanced (fig. 9-10).
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