CN106237318A - A kind of bacillus calmette-guerin vaccine dry powder micropin vaccine and preparation method thereof - Google Patents

Abstract

The invention discloses a BCG dry powder microneedle vaccine and a preparation method thereof. The microneedle is composed of BCG dry powder, a hollow microneedle array and a base patch, and the BCG dry powder is loaded in the hollow microneedle array. The preparation steps are as follows : (1) Cultivate BCG in 7H9 liquid medium, harvest the bacteria by centrifugation until the logarithmic growth phase, freeze-dry, and grind into powder; (2) Put 15% mass concentration of hyaluronic acid sodium salt into the microneedle mold , horizontally centrifuged and dried to prepare hollow microneedles; then BCG dry powder was filled into the microneedles, and 10% mass concentration of hyaluronic acid sodium salt was added to the surface of the mold, centrifuged, dried, and demolded. BCG dry powder can be stored in microneedles at room temperature for 90 days, with a large loading capacity and good immune effect. There is no need for professionals to operate, and no biological waste is generated.

Description

A kind of BCG dry powder microneedle vaccine and preparation method thereof

technical field

The invention belongs to the technical field of vaccines, and specifically relates to a BCG dry powder microneedle vaccine, and also relates to a preparation method of the dry powder microneedle vaccine, which can replace the traditional syringe intradermal vaccination method.

Background technique

Tuberculosis is currently the most harmful bacterial infectious disease. One-third of the world's people are infected, and 5% to 10% of them will develop clinical diseases sooner or later in their lifetime. According to the World Tuberculosis Report in 2015, there were about 9 million new cases and about 1.5 million deaths. The only vaccine currently licensed to prevent tuberculosis is Bacillus Calmette-Guerin (BCG). Bacillus Calmette-Guerin (BCG) has been around for 80 years and is one of the most widely used vaccines. In 1921, the BCG vaccine was first used for human immunity. In 1974, BCG was included in WHO's Expanded Immunization Program, and then the total immunization coverage rate reached over 80% in countries with high incidence of tuberculosis (TB). In countries that have included BCG in their childhood immunization programs, the coverage of BCG in newborns and infants has reached over 80%. BCG has a reliable protective effect against childhood meningitis and disseminated TB. Currently, approximately 100 million children receive BCG each year.

There are three ways of BCG vaccination: oral, scratch and subcutaneous injection. (1) Intradermal method: that is, intradermal injection. The injection site is at the outer edge of the lower end of the deltoid muscle of the left upper arm, and a mound should appear after the injection. For those with a negative tuberculin test, the vaccine used contains 0.5mg or 0.75mg of BCG per milliliter, and 0.1mL is used for intradermal injection, and subcutaneous injection is strictly prohibited. This method has a high and stable positive conversion rate. (2) Skin scratch method: The BCG used in the skin scratch is a milky white suspension containing 50-75 mg of bacteria per 1 mL. On the outer edge of the lower end of the deltoid muscle of the left upper arm, disinfect the skin with alcohol. After it dries, drop 2 to 3 drops of the shaken bacterin solution, and draw a "well" with a sterile needle, each 1 to 1.5 cm long, with a gap of 0.5 cm , It is advisable to have red marks, apply the vaccine evenly, let it penetrate into the skin, and wear clothes after the vaccine is dry. This method is simple to operate, easy to popularize and promote, with mild local reaction and less lymph node reaction. (3) Oral method: limited to babies within 2 months after birth. It has been clinically verified that the intradermal and oral methods have a higher positive conversion rate, while the skin scratch method has a lower positive conversion rate.

The WHO recommends intradermal inoculation, that is, injecting into the deltoid region of the upper arm with a syringe and needle. Although intradermal injection vaccination can produce better immune effect, there are also some unfavorable factors. First of all, due to the high requirements of vaccination technology, it needs to be operated by professionals; secondly, after injection and vaccination, the skin will produce redness, blisters, ulcers and other adverse reactions, and scars will be left. The inflammatory reaction will cause some infants to have fever; finally, Intradermal injection vaccination requires a syringe, which will form biological waste that needs to be disposed of, and the storage of BCG requires low temperature, which makes it inconvenient to inoculate BCG in some remote and backward areas.

The microneedle BCG vaccination technology has solved the adverse factors of the above-mentioned intradermal vaccination well. The current microneedle vaccines generally prepare microneedles by mixing vaccines with soluble materials. This preparation method can only deliver inactivated vaccines or proteins, and its storage time is short. Soluble polymer materials are immune to active biological macromolecules. The effects of origin are also difficult to assess. Hollow structure microneedles are mostly made of metal, silicon and other materials, and are generally used for the delivery of liquid preparations. Due to the difficulty in solving the problem of liquid leakage, they are rarely used in practice. The present invention utilizes soluble hollow microneedles as a BCG delivery tool, and directly delivers BCG (BCG) in the form of dry powder into the skin, thereby activating immune cells and achieving immune effects. The vaccine is delivered into the skin in the form of dry powder, which can preserve the physiological activity of the substance to the maximum extent and reduce the irritation to the skin. BCG is loaded in the microneedles in the form of dry powder, which greatly prolongs the storage period at room temperature, and greatly saves transportation and storage costs. In addition, BCG is directly sealed in the microneedles in the form of dry powder, so that the microneedles can be stored in the same size. The significant increase in the loading capacity makes it possible for BCG to immunize the human body with the immune patch, which overcomes the common problems of the current microneedle patch such as low drug loading and difficulty in meeting the actual clinical needs. The technology of the invention is simple to operate, no professional operation is required during immunization, and no biological waste is generated.

Contents of the invention

The object of the present invention is to provide a kind of BCG dry powder microneedle vaccine, based on the inoculation of the microneedle vaccine, it overcomes the adverse reactions such as redness, swelling, blisters, scars, inflammation and the like caused by the injection of BCG vaccine. In the microneedle structure, the drug loading can meet the clinical needs, and it can also be stored at room temperature for up to three months, which greatly saves transportation and storage costs, and does not require professional operation during inoculation, and no biological waste is generated .

Another object of the present invention is to provide a preparation method of BCG dry powder microneedle vaccine. Currently, the BCG vaccine is inoculated intradermally. The BCG freeze-dried powder needs to be stored at 4°C. In addition to bacteria, there are some other stable components in the vaccine. Before use, it is necessary to calculate the dilution amount and add the corresponding dilution solution to convert it into a liquid again. , and then draw the corresponding dose for infant vaccination. There are a series of possible error factors in these processes, leading to adverse reactions such as failure of immunization or exacerbation of inflammatory response. The microneedle BCG dry powder inoculation technology provided by the present invention optimizes the preparation of BCG. It only needs to directly collect the bacteria after cultivating BCG to the logarithmic growth phase, and then wash with PBS several times without adding any auxiliary agent, directly vacuum freeze-drying, and then Grind it into a uniform powder. This preparation method not only simplifies the operation steps of BCG, shortens the production time, but also saves the production cost.

In order to achieve the above object, the present invention adopts the following technical solutions:

A BCG dry powder microneedle vaccine, characterized in that it consists of BCG dry powder, a hollow microneedle array and a base patch, the BCG dry powder is loaded in the hollow microneedle array, and the composition of the hollow microneedle array is 15% Mass concentration of hyaluronic acid sodium salt, the composition of the base patch is 10% mass concentration of hyaluronic acid sodium salt.

A preparation method of BCG dry powder microneedle vaccine, the steps are:

1. Preparation of Bacillus Calmette-Guerin dry powder: Bacillus Calmette-Guerin (BCG) was cultivated in 7H9 liquid medium, and the bacteria were harvested by centrifugation in the logarithmic growth phase, washed 3 times with phosphate-buffered saline (PBS), freeze-dried, and ground into powder;

2. Filling of BCG dry powder:

(1) Put 15% (w/v) hyaluronic acid sodium salt (HA) in the microneedle mold, and centrifuge horizontally at 1500rpm at 4°C for 10min; take out the mold and rotate it 180 degrees, and centrifuge horizontally at 2200rpm at 4°C for 20min; Take out the mold and rotate it at 180 degrees, and then centrifuge horizontally at 3000rpm at 4°C for 30 minutes. Through the above operations, the sodium hyaluronic acid salt can fully enter the microneedle mold to form needles; remove the excess sodium hyaluronic acid salt on the surface of the mold, and form a hollow structure after drying microneedle array;

(2) Pour the BCG dry powder into the prepared microneedle hollow structure, centrifuge horizontally at 3000rpm at 4°C for 10 minutes, take out the mold and rotate it 180 degrees, and continue to centrifuge horizontally for 10 minutes, and repeat the centrifugation for 3 times so that the powder enters the hollow part of the microneedle , and remove excess dry powder on the surface of the mold;

(3) Add 10% (w/v) sodium hyaluronic acid on the surface of the mold, and centrifuge at 3000rpm at 4°C for 10min; take out the mold and rotate it at 180 degrees, and continue to centrifuge at 3000rpm at 4°C for 10min; take out the mold, dry it, and remove it. mold.

Preferably, the height of the microneedles is 200-500 μm.

Compared with the prior art, the present invention has the following advantages:

1. The current BCG needs to be stored in a 4°C refrigerator, which increases the cost of storage and transportation. When used, it needs to be re-diluted in proportion, and then injected by a professional; the microneedle patch used in the present invention seals the BCG in a micro In the hollow structure of the needle, it can be stored at room temperature for more than 3 months. It is easy to use and operate. You only need to press the microneedle patch into the skin. You can inoculate yourself without professional operation, and you don’t need other auxiliary diluents and syringes. , is a "ready-to-use" vaccine.

2. Another advantage of the present invention is that the microneedle BCG dry powder immunization has no skin irritation reaction. The traditional intradermal injection vaccination is marked by the occurrence of "scars". More than 95% of infants and young children will have redness, swelling, ulceration, and scars after vaccination. Some infants also have adverse side effects such as fever and lymph node enlargement. occur. In the present invention, the BCG is subpackaged in the microneedle array, which is equivalent to multi-point simultaneous micro-immunization, without any skin irritation, pain, and no scars, and reduces the psychological rejection of family members' adverse effects of BCG vaccination.

3. A series of experimental comparisons show that microneedle BCG dry powder patch immunization has the same effect as traditional intradermal immunization; it is better than traditional intradermal immunization in stimulating NK cell activity. It is speculated that multi-point micro-immunization may be better Activates the innate immune response.

4. Compared with the existing BCG preparations, the preparation method of microneedle BCG dry powder is simple. It only needs to collect the bacteria, wash them with PBS, freeze-dry them, and then grind them into powder. There is no need to prepare diluent for later use. In addition to bacteria, the dry powder does not contain any culture solution or protective agent, which further reduces the possibility of skin irritation.

5. The microneedle used in the present invention is made of soluble material HA and has a hollow structure. The early microneedles mainly used solid microneedles made of insoluble materials, which may break when used, causing the needles to stay in the skin, which brings inestimable dangers; insoluble materials such as glass can also be used to prepare hollow needles. Microneedles are used to inject liquid preparations. The front ends of the microneedles have openings, which can easily cause liquid leakage, making it difficult to accurately evaluate the amount of drug delivered. Preparation of microneedles from soluble materials is the current mainstream. When preparing microneedles from soluble materials to deliver drugs, the drug is generally mixed with a polymer and then prepared by polymerization. The dissolution of the microneedles is carried out simultaneously with the release of the drug. Needles are not suitable for carrying attenuated vaccines or active biomolecules, and the load capacity is limited. The microneedle used in the present invention is not only made of soluble materials, but also has a hollow structure, which can carry any powdery substance, including active vaccines and proteins, etc., has a long storage period and a large load, and is suitable for clinical promotion.

Description of drawings

Figure 1 is a comparison of hollow microneedles and microneedles loaded with BCG dry powder. The upper layer is a hollow microneedle, and the lower layer is a microneedle loaded with BCG dry powder and an observation picture of its bottom.

Figure 2 is a picture of microneedles loaded with SRB-stained BCG dry powder under a confocal microscope.

Fig. 3 is the maximum absorption wavelength of the emitted fluorescence of SRB-BCG detected by the microplate reader.

Fig. 4 is a standard curve of fluorescence absorption value and weight of SRB-BCG at a wavelength of 640nm.

Figure 5 shows the detection of SRB-BCG dry powder loading. The content of BCG dry powder in 9 microneedles was randomly detected, the experiment was repeated three times, and 6 microneedle patches were detected each time.

Figure 6 is the determination of the shelf life of microneedle BCG. Taking the day when the microneedle BCG was prepared as "0" day, store it in a dry and light-proof environment for 30 days, 60 days, and 90 days. After that, 24 needles were randomly cut from the microneedle patch, dissolved in PBS, and counted by plate counting method. Count (n=6).

Fig. 7 is the detection result of skin inflammation after inoculation in Example 3. ID represents the intradermal injection group, PBS represents the injection control group, MN empty needle control group, and BCG-MN represents the microneedle BCG immunization group. Among them, 7-A is the skin reaction at the inoculation site, redness, swelling, blisters, and scars appeared in the ID group, and there was no skin reaction in the MN group and BCG-MN group, and the injection needle hole could be seen on the first day and the third day in the PBS group; 7 -B is the HE staining microscopic image of the inoculated skin on the third day after inoculation, a large number of inflammatory cell infiltration can be seen in the ID group, but no inflammatory cells are produced in the BCG-MN group; In other test groups, the skin temperature at the inoculation site increased significantly on days 2, 3, 4, 5, 6, 7, and 8 (*P<0.05, **P<0.01, ***P<0.001).

Fig. 8 is the detection result of NK cell and T cell immune response after vaccination. Among them, A is the detection of IL-2 activity in NK cells in peripheral blood one week after immunization, and it is found that the amount of IL-2 secreted by NK cells in the BCG microneedle immunization group (BCG-MIN) is significantly higher than that in the MN group and PBS group; B is the immunization Peripheral blood was collected in the next week to detect the activity of TNF-α in NK cells, and it was found that the secretion of TNF-α in NK cells had no significant difference in each group; Factor IFN-γ activity, it was found that the secretion of IFN-γ in ID group and BCG-MN group was significantly higher in CD4 ⁺ T cells (C) and CD8 ⁺ T cells (D) than in MN group and PBS group, BCG-MN The value of IFN-γ in group IFN-γ was slightly higher than that in ID group, but there was no significant difference; E and F were collected peripheral blood at the 4th week after immunization to detect TNF ^- α in T cells. -The amount of secreted TNF-α in MN group was significantly higher than that in MN group and PBS group (E), but there was no difference in CD8 ⁺ T cells (F), *P<0.05, NS: no significant difference.

Figure 9 shows the detection results of antibodies (IgG, IgG1, gG2a) after 4 weeks of immunization. Among them, A, B, and C are the detection results of IgG, IgG1, and gG2a respectively. The results show that there is no significant difference in the three kinds of antibodies produced by the ID group and the BCG-MN group (BCG dry powder immunization group), *P<0.05,** *P<0.001.

Figure 10 shows the detection results of spleen and lung cytokines 3 months after immunization. Among them, A-D are the detection results of spleen cytokines TNF-α, IL-6, IL-12 and IL-17, respectively, and E-H are the detection results of lung cytokines TNF-α, IL-6, IL-12 and IL-17, respectively . The results showed that the BCG-MN group (BCG dry powder immunization group) and the ID group could significantly stimulate the production of corresponding cytokines, and there was no significant difference between the BCG-MN group and the ID group. *P<0.05, **P<0.01, ***P<0.001.

detailed description

The present invention establishes a new microneedle patch vaccine for BCG vaccination, and the detailed steps are as follows:

Example 1

The preparation method of BCG dry powder microneedle vaccine, its steps are:

1. Cultivate BCG (ATCC-35733, other BCG vaccine strains) in 7H9 liquid medium (BBL ^TM Mycoflasic Middlebrook 7H9broth with glycerol prepared media, BD) at 37°C for 4 weeks; centrifuge at 10,000rpm for 5min, and collect the bacteria , washed 3 times with phosphate-buffered saline (PBS), resuspended in PBS after centrifugation, pre-cooled at 4°C for 2h, then pre-cooled at -20°C for 2h, and frozen at -76°C for 2h, then placed in Dry it in a freeze dryer, all of the above are aseptic operations; pour the BCG dry powder into a sterilized grinder and grind it thoroughly, then pour the ground bacteria into a sterile storage tube, and store it sealed at 4°C for later use.

2. Prepare microneedle by two-step method: 50 μ L of 15% (w/v) hyaluronic acid sodium salt (HA) is placed in microneedle polydimethylsiloxane (PDMS) mold, and the mold specification The size is 1cm×1.3cm, the microneedle height is 200-500μm, 4°C 1500rpm horizontal centrifugation (centrifuge Thermo GP8R) for 10min; the mold is taken out and rotated 180 degrees, and 4°C 2200rpm horizontal centrifugation for 20min; the mold is taken out and rotated 180°placed, Centrifuge horizontally at 3000rpm at 4°C for 30 minutes. Through the above operations, sodium hyaluronic acid can fully enter the microneedle mold to form a needle; wipe off excess sodium hyaluronic acid on the surface of the mold with a cotton swab, collect it for later use, and place the mold in In a negative pressure dryer, dry overnight at room temperature to form a microneedle array with a hollow structure; then pour 2mg of BCG dry powder into the prepared hollow microneedle structure, centrifuge horizontally at 3000rpm at 4°C for 10min, take out the mold and rotate it 180 degrees Place it, continue to centrifuge horizontally for 10min, repeat centrifugation for 3 times like this, make the powder enter the hollow part of the microneedle, wipe off the excess powder on the surface with a cotton swab; add 10% (w/v) sodium hyaluronic acid ( HA) 30μL, centrifuge at 3000rpm at 4°C for 10min; take out the mold and rotate it 180 degrees, and continue to centrifuge at 3000rpm at 4°C for 10min; then take out the mold, place it in a desiccator for 3h at room temperature, and take it out with tweezers after drying, that is, the BCG is filled Dry powder microneedle patch.

Example 2

BCG dry powder load and shelf life determination in BCG dry powder microneedle vaccine, its steps are:

1. Collect BCG according to the method described in step 1 in Example 1, wash with 1 mL of PBS and resuspend, add 0.5 g of sulforhodamine B (SRB) powder, wash with PBS several times after staining for 10 min, until the washing liquid is colorless Then make dry powder according to the method described in step 1 in Example 1, and make the dyed BCG microneedle patch with a height of 200 μm according to the method described in step 2; take 1 mg of dyed BCG dry powder and dilute with PBS , measure the absorbance at 640nm, and make a standard curve; in the prepared 6 microneedle patches loaded with dyed BCG dry powder, cut 9 microneedles from each microneedle patch and dissolve them in PBS, respectively, at 640nm For colorimetry, the average load of a single needle was calculated according to the standard curve, and the experiment was repeated 3 times.

The experimental results show (see FIG. 5 ) that the load of 9 needles is between 2.2-2.3 μg, the average load of a single needle is 0.25 μg, and the confidence interval is 95%.

2. Prepare 24 microneedle patches loaded with BCG dry powder according to the method described in Example 1, and each 6 pieces is a group, divided into 4 groups for storage, and the day of preparation is 0 days, and they are stored for 30 days and 60 days respectively. 90 days; 24 microneedles were cut from 6 patches each time, dissolved in 200 μL PBS, after doubling dilution, the solution was spread on 7H10 agar plate (BD, Deep Fill), each gradient 3 Plates were cultured at 37°C for 30 days and counted.

The results showed that after the microneedle BCG dry powder was stored at room temperature for 90 days, the number of viable bacteria did not decrease significantly.

Example 3

Detection of skin inflammation after inoculation with BCG dry powder microneedle vaccine, the steps are

1. Divide 5-week-old BALB/c mice into 4 groups: syringe intradermal injection group (ID), BCG dry powder microneedle patch group (BCG-MN), PBS control group (PBS) and microneedle empty needle patch group Sheet control group (MN), with 7 mice in each group; the mice in each group were depilated with depilatory cream after anesthesia, each mouse in the intradermal injection group was injected with 6 μg/20 μL of BCG bacterial liquid, and each mouse in the BCG dry powder microneedle patch group was Only 24 needles/tablet were pressed in, and 20 μL PBS was injected intradermally in the PBS group, and 24 needles/tablet not filled with BCG were pressed into each mouse in the empty needle group.

2. Detect the temperature change of the mouse skin inoculation site every day after inoculation, and take pictures of the inoculation site on the 1st, 3rd, 5th, and 10th day to observe the inflammatory reaction on the skin surface.

The results showed (see Figure 7A), redness and swelling occurred in the ID group on day 1-3, blisters appeared on the fifth day, and scars appeared after the blisters disappeared, and the scars could still be observed until the tenth day; while the MN group and the BCG-MN group had no In case of any skin reaction, the injection needle hole could be seen in the PBS group on the 1st and 3rd day, and then healed without visible skin reaction. In each experimental group, one mouse was randomly taken out from each group on the 3rd day, and the skin of the inoculation site was peeled off after being sacrificed. After being fixed with paraformaldehyde (4%), tissue sections were prepared, and pathological changes were observed by HE staining. HE staining microscopic examination of tissue sections revealed that a large number of inflammatory cell infiltrations could be seen in the ID group, while no inflammatory cells were produced in the BCG-MN, MN and PBS groups (Fig. 7B). The skin temperature monitoring showed that compared with other test groups, the skin temperature at the inoculation site in the ID group increased significantly on the 2nd, 3rd, 4th, 5th, 6th, 7th, and 8th day, which also indicated a strong skin inflammatory reaction in the ID group (Figure 7C) .

Example 4

The immune effect test after inoculation with BCG dry powder microneedle vaccine, the steps are:

1. Cytokine detection

The experimental subject is the mouse treated by the method described in step 1 in Example 3, and is processed according to the following steps in the 1st week and the 4th week respectively:

1. Mouse orbital blood collection: Add 10 μL of heparin (to prevent blood coagulation) into the EP tube, insert a capillary into the peripheral venous sinus of the orbit of the anesthetized mouse, drain the blood into the EP tube, and take 200 μL for each mouse; The EP tube was centrifuged directly (4°C, 3500rpm, 5min), and the upper layer of serum was carefully sucked away with a pipette gun (20μL), and the blood cells were retained; the blood cells were resuspended in 100μL of PBS, transferred to the flow tube (100μL), and then disrupted. Red agent 1.5-4mL; place on ice for 5min, centrifuge at 1800rpm at 4°C for 3min; after centrifugation, pour off the supernatant, and scrape the flow tube on the test tube rack to facilitate the collection of precipitates; add 1mL PBS to wash, the same Centrifuge for 5 minutes under centrifugation conditions, remove the supernatant, and repeat washing once; add 1 mL of cell culture medium (containing 10% FBS and P/S double antibody), centrifuge and wash according to the above conditions, discard the supernatant; add 100uL of medium to each tube After mixing, the cells were transferred to a 96-well cell culture plate; stimulated with stimulatory proteins (ESAT-6, Ag85, CFP-10 and TB 10.4) at a final concentration of 2 μg/mL, cultured overnight at 37°C; Add 1 μL of blocking agent (Golgiplug, 1:1000), and continue culturing for 5 hours.

2. Blow up the cells in the 96-well plate again, transfer them to the flow tube, mark them well, wash them twice with 1 mL of PBS containing 2% FBS, and the conditions are the same as above.

3. Add 1 μL of the following surface marker antibodies respectively, and collect blood to detect the secretion of NK cell (CD49b-PE) internal factor IL-2 (IL-2–PE-cy7) in the first week after immunization; collect blood to detect T Cellular (CD4-FITC, CD8-Percp-cy5.5) secretion of intrinsic factors IFN-γ, TNF-a and IL-2 (IFN-γ-FITC, IL-2–PE-cy7, TNF-a-APC) ; Put it on the test tube rack and wrap it with tin foil, let it stand at room temperature for 30min, and wash it twice with 1mL LPBS. Add 200 μL of fixative solution, put it in a 4°C refrigerator for 20 minutes, then add 1 mL of 1×wash solution (BD), leave it at room temperature for 15 minutes, centrifuge to remove the supernatant; repeat it again; add 100 μL of wash solution to each sample, add each Each antibody (IFN-γ, TNF-α, IL-2) 1 μL, placed in a 4 ℃ refrigerator, wrapped in tin foil and incubated for 30 minutes, then washed twice with 1 mL of wash solution according to the above operation, resuspended in 200 μL of wash solution, wrapped in tin foil , detected by flow cytometry.

The results of cytokine detection (see Figure 8) showed that, like the ID group, the BCG-MN group could effectively stimulate the activation of T cells, and was superior to the ID group in stimulating the activation of NK cells.

2. Antibody detection

1. After 4 weeks of immunization, blood was collected according to the method described in step 1 above, and serum was collected after centrifugation.

2. Collect the BCG culture, centrifuge at 17700g for 30min at 4°C; wash the bacteria twice with PBS, resuspend the pellet in a solution containing sodium deoxycholate [5-15% (w/v), 0.1-0.25mL/ g] buffer [30mM Tris, 2mM EDTA, pH 8.5]; after 1h, the sample was centrifuged again according to the above method, and the supernatant was collected, and then the supernatant was centrifuged at 70,000g for 4h at 4°C in an ultracentrifuge; Supernatants, pellets were resuspended in buffer [30 mM Tris, 2 mM EDTA, pH 8.5], filtered with filters [0.45 μm and 0.2 μm] and stored at 4°C until use.

3. Coat 100 μL of the BCG antigen extracted in the above step 2 on an ELISA plate, block with skim milk for 2 hours, add HRP cross-linked goat anti-mouse IgG (NA931V, GE healthcare, dilution 1:6,000), IgG1 (A90-105P ,Bethyl,dilution 1:10,000), IgG2a(61-0220,Life Technologies,dilution 1:2,000), 37°C for 1h, wash 3 times, add 50μL of chromogenic solution and react in the dark for 20min, then add stop solution 4% 50 μL of H ₂ SO ₄ , and detect the absorbance at 490 nm with a microplate reader.

The experimental results showed (see FIG. 9 ), that the BVG-MN group, like the ID group, could effectively stimulate the host to produce specific antibodies.

3. Spleen and lung cytokine detection

1. Experiment preparation: take a 12-well culture plate, fill it with 2mL DEME culture solution, place it on ice, after the mouse is anesthetized, spray the surface with 70% alcohol to sterilize, place it on a foam board, and lavage the lungs with PBS; take the lungs , spleen, placed in a 12-well culture plate;

2. Lung treatment: Add 0.5mL of ice-bathed CD solution (HBSS buffer [no calcium and magnesium, containing phenol red]+150U/mL collagenase+10U/mL DNAse II) into the EP tube, put the lungs in, repeat Cut into pieces, then add 0.5mLCD and 2.5μL calcium chloride (0.8mM), put it in a 37°C water bath, vortex every 15min, digest for 1h; pour all into a small plate with medium into a 70μm filter Grind, rinse the EP tube with 1mL LPBS, and pour into the strainer. After grinding, wash the filter screen once with 1mL PBS, transfer it all to a 50mL centrifuge tube, put a filter membrane (40μm filter membrane) at the mouth of the tube, wash the filter screen with 1Ml PBS; collect the filtrate, put it into a centrifuge at 4 degrees 1500rpm for 3min ; Remove the supernatant after centrifugation, add 4mL ACK to break the red in an ice bath for 10min. Wash the pellet twice with 10 mL PBS; after washing the pellet, resuspend in 1 mL DMEM medium, drop 10 μL of trypan blue on the side of the cell counting plate, take 10 μL of cell solution, mix well, count the cells, and adjust the cell concentration (2× 10 ⁶ cells/mL); transferred to culture medium (DMEM plus 10% FBS and 100U/mL penicillin and 100mg/mL streptomycin), 96-well plate, each plus 200μL cell culture medium (or 12-well plate, each Well plus 1mL).

3. Spleen treatment: After the spleen cells are taken out, put them in a 12-empty plate, ice-bath with an appropriate amount of medium, and then grind them in a 70 μm filter, rinse the filter with 1 mL of LPBS; collect the filtrate and filter it with a 40 μm filter Transfer to a 50mL centrifuge tube, rinse the filter with 1mL PBS; centrifuge at 4 degrees at 1500rpm for 3 minutes, discard the supernatant, add 4 mL of ACK to break the red and bathe in ice for 10 minutes (after breaking the red, there will always be flocculent tissue precipitation, so it can be considered to break the red first, and then filter ); centrifuge at 1500 rpm at 4°C for 3 minutes, wash twice with 10 mL of PBS, add 2 mL of DMEM medium to resuspend splenocytes, and count. Experience shows that the number of viable cells obtained is about 2x10 ⁶ /mL.

4. Adjust spleen and lung cells to 2x10 cells/mL, transfer 200 μL each to a 96 ^- well plate (or 1 mL to a 12-well plate), and mark according to the amount of ELISA required.

5. Add stimulatory proteins according to the method described in Example 4 (final concentration of various stimulatory proteins: 2ug/ml) to stimulate cell culture for 72 hours, centrifuge to get the supernatant, put it in a refrigerator, and use the corresponding ELISA kit (eBiosciences) Detection and analysis of cytokines.

The experimental results showed (see FIG. 10 ), both the BCG-MN group and the ID group could significantly stimulate spleen and lung cells to produce TNF-α, IL-6, IL-12 and IL-17 factors.

The above series of experiments confirmed that the BCG dry powder microneedle patch vaccination technology has an immune effect similar to that of the traditional syringe intradermal vaccination, and is superior to the traditional intradermal injection vaccination method in terms of stimulating the immune effect of NK cells. In addition, in terms of skin irritation, vaccine transportation and storage, it has incomparable advantages over the intradermal injection method.

Claims (3)

1. a bacillus calmette-guerin vaccine dry powder micropin vaccine, it is characterised in that by bacillus calmette-guerin vaccine dry powder, the microneedle array of hollow and substrate paster Composition, bacillus calmette-guerin vaccine dry powder is loaded in the microneedle array of hollow, and the composition of described hollow microneedle arrays is 15% mass concentration Hyaluroni, the hyaluroni that composition is 10% mass concentration of described substrate paster.

Bacillus calmette-guerin vaccine dry powder micropin vaccine the most according to claim 1, it is characterised in that: the height of described micropin is 200- 500μm。

3. the preparation method of the bacillus calmette-guerin vaccine dry powder micropin vaccine described in claim 1, the steps include:

(1) preparation of bacillus calmette-guerin vaccine dry powder: be incubated at by bacillus calmette-guerin vaccine in 7H9 fluid medium is thin to exponential phase harvested by centrifugation Bacterium, washs 3 times by phosphate buffered solution, lyophilization, grind into powder；

(2) filling of bacillus calmette-guerin vaccine dry powder:

A) hyaluroni of 15% mass concentration is placed in micropin mould, 4 DEG C of 1500rpm horizontal centrifugal 10min；By mould Tool takes out rotation turnback and places, 4 DEG C of 2200rpm horizontal centrifugal 20min；Mould takes out rotation turnback place, 4 DEG C 3000rpm horizontal centrifugal 30min；Remove the hyaluroni that die surface is unnecessary, the dried micropin forming hollow structure Array；

B) bacillus calmette-guerin vaccine dry powder is poured in the micropin hollow structure prepared, 4 DEG C of 3000rpm horizontal centrifugal 10min, mould is taken Go out to revolve turnback to place, continue horizontal centrifugal 10min, be so centrifuged repeatedly 3 times, make powder enter micropin hollow space, and go Except the dry powder that die surface is unnecessary；

C) add the hyaluroni of 10% mass concentration at die surface, 4 DEG C of 3000rpm are centrifuged 10min；Mould is taken Going out to revolve turnback to place, 4 DEG C of 3000rpm continue centrifugal 10min；Mould is taken out, is dried, the demoulding.