CN110680909A - Quick-release type-b haemophilus influenzae conjugate vaccine soluble microneedle patch and preparation method thereof - Google Patents

Abstract

The invention provides a quick-release b-type haemophilus influenzae conjugate vaccine soluble microneedle patch which comprises a needle body and a basal layer, wherein the needle body consists of b-type haemophilus influenzae conjugate vaccine, an adjuvant and a water-soluble high polymer material, and the basal layer is a flexible high polymer material; the content ratio of the adjuvant to the Haemophilus influenzae type b conjugate vaccine is (1: 1) - (2: 1), and the adjuvant is one or more of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A. The soluble microneedle patch of the haemophilus influenzae type b conjugate vaccine can balance Th1/Th2 immune response and quickly release the vaccine in skin tissues.

Description

Quick-release type-b haemophilus influenzae conjugate vaccine soluble microneedle patch and preparation method thereof

Technical Field

The invention relates to the technical field of microneedle administration of vaccines, and provides a quick-release b-type haemophilus influenzae combined vaccine soluble microneedle patch and a preparation method thereof.

Background

A Haemophilus influenzae type b (Hib) conjugate vaccine is prepared by covalently binding purified Haemophilus influenzae type b capsular polysaccharide with tetanus toxoid (or other carrier proteins). The haemophilus influenzae type b is a common symbiotic bacterium in nasopharynx of children, most children carry Hib in a certain period and sometimes carry Hib for months before the vaccine is applied, but the bacterial colonization rate is greatly different from the social and economic conditions according to age. In areas with high Hib vaccination rate, the colonization rate of bacteria in the nasopharynx is very low due to the group immunization effect of the Hib conjugate vaccine. Although only a small percentage of carriers will develop into clinical cases, carriers are of great significance in the transmission of disease as Hib is transmitted by droplets of respiratory secretions.

Hib disease is primarily manifested as pneumonia and meningitis in children, and is a major public health problem in areas where large-scale Hib vaccination has not been performed. According to the 2006 WHO announces that there are at least 300 million serious cases worldwide per year, about 38.6 million deaths. Hib morbidity and mortality are mostly seen in developing countries, and the disease burden is most severe in children at 4-18 months of age, but occasionally morbidity occurs in children less than 3 months of age and children more than 5 years of age. In non-immune populations, Hib is the leading cause of non-epidemic bacterial meningitis in children under the age of 1 year. Even if sufficient antibiotic therapy is given in time, 3-20% of Hib meningitis patients still die. In areas with insufficient medical and health resources, the death rate of Hib meningitis is higher, and the nervous system sequelae in survivors are common and account for about 30-40%. Other important but uncommon clinical manifestations of Hib infection exist: sepsis, suppurative arthritis, osteomyelitis, pericarditis, cellulitis and epiglottitis (especially in industrialized countries). An aggressive Hib disease is established if Hib bacteria are detected in normally sterile body fluids or tissues such as blood, cerebrospinal fluid, abdominal fluid, thoracic fluid or lung aspirates. The Haemophilus influenzae type b conjugate vaccine is generally administered through intramuscular injection, and generally comprises water injection and powder injection. However, the injection usually needs the supervision of a doctor, the injection needle can stimulate nerve cells in the deep layer of the skin, which is easy to cause pain to the human body, the patient compliance is poor, and the infection risk exists.

The microneedle transdermal immunization approach is a novel transdermal immunization (TCI) method, and the method has been developed for decades from the time of presentation to the time of continuous development, and is characterized in that the epidermal horny layer can be punctured, so that a microchannel is created, which is beneficial for vaccines to enter dermis and subcutaneous tissue, and then the vaccines are absorbed through capillary vessels to improve bioavailability, painless administration can be realized, the compliance of patients is good, and the operation is simple and convenient. The micro-needles can be divided into four types, namely solid insoluble micro-needles, coated micro-needles, soluble micro-needles and hollow micro-needles according to the structure and the drug release mode of the micro-needles. The soluble microneedle can be dissolved automatically after penetrating into human skin, avoids the problems of breakage of a solid microneedle head, small drug-loading rate of a coated microneedle and the like, is suitable for delivering water-soluble and other biological macromolecules, and has a good application prospect.

Chinese patents CN201310071361.0 and CN201620500195.0 provide a sustained release soluble microneedle and a preparation method thereof, respectively, but the technology is not suitable for vaccines with low vaccination times and vaccines which need to be released immediately after vaccination.

The Chinese patent with the application number of CN201710854856.9 provides a quick-release soluble microneedle and a preparation method thereof, but the technology does not add corresponding adjuvants, so that the immunization effect is poor and more antigen vaccines are wasted.

The adjuvant is a substance which can obviously enhance the immunogenicity of an antigen after being added into a vaccine and has no immunogenicity, and can obviously enhance the immune effect of the vaccine and reduce the dosage of the antigen when being applied to the haemophilus influenzae type b conjugate vaccine, thereby being beneficial to the improvement of the production efficiency and the reduction of the production cost of the haemophilus influenzae type b conjugate vaccine, expanding the number of inoculated groups, better protecting susceptible groups and reducing the huge pressure brought by the vaccine production enterprises due to insufficient yield. The main adjuvants which are widely applied at present are: aluminum adjuvants (aluminum oxide, aluminum hydroxide, aluminum phosphate), MF59 adjuvant, and the like.

It is well known that most vaccines must be combined with adjuvants to exert effective immunoprotection, and that not all combinations of adjuvants and vaccines produce significant improvements in immunoprotection. For example, it has been shown that transdermal immunization of vaccine microneedles tends to induce a stronger Th 2-type humoral immune response, but balancing the Th1/Th 2-type immune response is more important for invasive diseases caused by Haemophilus influenzae (see, e.g., Kim Y C, Yoo D G, CompsR W, et al. Cross-protection by co-immunization with underfluorinated virus vaccine DNA and inactivated virus vaccine used coated microrodeles. journal of controlled Release,2013,172(2): 579-.

Therefore, how to select a proper adjuvant to be added into a soluble microneedle patch of the haemophilus influenzae type b conjugate vaccine to develop a soluble microneedle capable of releasing drug in skin tissues quickly, and realize the balance of Th1/Th2 type immune response and improve the transdermal immune effect is a technical problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

The invention aims to provide a quick-release haemophilus influenzae type b conjugate vaccine soluble microneedle patch and a preparation method thereof, which solve the bottleneck of the prior art.

In order to realize the research and development thought, the technical scheme adopted by the invention is as follows:

a quick-release b-type haemophilus influenzae conjugate vaccine soluble microneedle patch comprises a needle body and a basal layer, wherein the needle body is composed of a b-type haemophilus influenzae conjugate vaccine, an adjuvant and a water-soluble high polymer material, and the basal layer is a flexible high polymer material; the content ratio of the adjuvant to the Haemophilus influenzae type b conjugate vaccine is (1: 1) - (2: 1), and the adjuvant is one or more of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A.

Preferably, the water-soluble polymer material is a mixture of PVP and Gantrez, wherein the content ratio of the PVP to the Gantrez is 3: 1.

Preferably, the adjuvant is a mixture of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A, and the content ratio of the three is SPO 1: astragalus polysaccharide: 3-de-O-acylated monophosphoryl lipid a ═ (1: 1: 2) - (1: 2: 1).

Preferably, the needle body further comprises a heat-resistant protective agent.

More preferably, the heat-resistant protective agent is trehalose, and the flexible high polymer material is hydroxypropyl methylcellulose.

Preferably, the needle body consists of a haemophilus influenzae type b conjugate vaccine, an adjuvant, a water-soluble high molecular material and trehalose, wherein the content ratio of the adjuvant to the haemophilus influenzae type b conjugate vaccine is 1: 1, adjuvant is 1: 2: 1 of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A, and the water-soluble high molecular material is the mixture of PVP and Gantrez with the content ratio of 3: 1; the basal layer is hydroxypropyl methylcellulose.

The invention also provides a preparation method of the quick-release haemophilus influenzae type b conjugate vaccine soluble microneedle patch, which comprises the following steps:

1) preparing a needle body solution: dissolving the water-soluble high polymer material, the haemophilus influenzae type b conjugate vaccine and the adjuvant with water according to the prescription amount, and uniformly mixing to obtain a needle body matrix solution;

2) preparation of base layer solution: dissolving a prescription amount of flexible high polymer material by using water to obtain a substrate solution;

3) preparation of soluble microneedles: injecting the needle body solution into the microneedle female die, centrifuging at 4000rpm for 20min, scraping off the redundant needle body solution, drying in a normal-temperature drying oven for 1h, adding the base layer solution, centrifuging at 4000rpm for 20min again, drying the microneedle female die at normal temperature for 24h, and demolding to obtain the microneedle female die.

In the invention, the shape of the microneedle body can be one or more of conical shape, polygonal pyramid shape, columnar shape and the like, and is preferably conical; the diameter of the bottom of the needle part is 100-500 mu m; the height of the needle body is 100-2000 μm.

Compared with the prior art, the quick-release haemophilus influenzae type b conjugate vaccine soluble microneedle patch provided by the invention has the innovativeness that:

the microneedle adopts the composition of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A with a proper proportion as an adjuvant, has a remarkable immunity enhancement effect compared with a classical aluminum adjuvant, can effectively improve the antibody level of a vaccine antigen, the secretion level of cell factors and other humoral immunity and cellular immunity levels, and realizes balanced Th1/Th2 type immune response. In addition, the micro-needle is prepared from a soluble biodegradable material, so that the haemophilus influenzae type b conjugate vaccine can be quickly released in skin tissues.

Drawings

FIG. 1 is a schematic structural diagram of a soluble microneedle of a Haemophilus influenzae type b conjugate vaccine of the present invention.

FIG. 2 is a flow chart of the preparation of soluble micro-needles of the Haemophilus influenzae type b conjugate vaccine of the present invention.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the technical solution in the present embodiment will be specifically described below with reference to the accompanying drawings in the present application. It should be noted that the following examples are only for illustrating the present invention and are not to be construed as limiting the present invention, and any modifications and changes made to the present invention within the spirit and scope of the claims are included in the scope of the present invention.

Example 1: preparation of soluble microneedle patch of non-adjuvant-added haemophilus influenzae type b conjugate vaccine

The preparation method comprises the following steps:

1) preparing a needle body solution: the water-soluble polymer material (for example, PVP and Gantrez, but not limited thereto, in a content ratio of 3: 1) and haemophilus influenzae type b conjugate vaccine are dissolved in water to prepare a needle body base solution.

2) Preparation of base layer solution: the flexible polymer material (for example, but not limited to hypromellose) is dissolved in water to form a base solution.

3) Preparation of soluble microneedles: injecting the needle body solution into a microneedle female die, centrifuging at 4000rpm for 20min, scraping off the redundant needle body solution, drying in a normal-temperature drying oven for 1h, adding the base layer solution, centrifuging at 4000rpm for 20min again, drying the microneedle female die in the normal-temperature drying oven for 24h at normal temperature, and demolding to obtain the b-type haemophilus influenzae conjugate vaccine soluble microneedle.

TABLE 1 prescription of different needle body matrix material ratios

A b-type haemophilus influenzae conjugate vaccine soluble microneedle patch was prepared according to the prescription of different needle body matrix material ratios in table 1 above, and the microneedle patch intradermal dissolution test performed in example 2 below was used to screen the water-soluble polymer material and ratio of the microneedle body.

Example 2: comparative intracutaneous dissolution Performance of soluble microneedle patch prepared in example 1

After the b-type haemophilus influenzae conjugate vaccine soluble microneedle is prepared according to different prescriptions in example 1, the microneedle is pressed into the skin of 6 different rats by using a thumb, the microneedle is pulled out at the time points of 0.2min, 0.5min, 1min, 2min, 3min, 4min, 5min, 6min, 7min and the like, and the intracutaneous dissolution condition of the microneedle is observed through a microscope. The results are shown in Table 2.

Table 2 intradermal dissolution of each microneedle (n ═ 6)

As can be seen from table 2, formula 2 has a better dissolution rate in microneedle intradermal than other formulas, so the mixture of PVP and Gantrez is selected as the water-soluble material in the needle body, and the content ratio of the two is 3: 1.

example 3: preparation of adjuvant-containing b-type haemophilus influenzae conjugate vaccine soluble microneedle patch

With reference to examples 1 and 2, the following procedure was used:

1) preparing a needle body solution: the water-soluble high polymer material PVP and Gantrez (calculated according to the content ratio of 3: 1), the Haemophilus influenzae type b conjugate vaccine, the heat-resistant protective agent (taking trehalose as an example, but not limited to the trehalose) and the adjuvant (see the adjuvant prescription in the table 3) are dissolved by water to be used as a needle body matrix solution. In this embodiment, in order to eliminate unnecessary influence factors, the contents of the water-soluble polymer material and the heat-resistant protective agent are limited to fixed values, so as to facilitate statistics, but not limited thereto.

2) Preparation of base layer solution: the flexible polymer material (for example, but not limited to hypromellose) is dissolved in water to form a base solution.

3) Preparation of soluble microneedles: injecting the needle body solution into a microneedle female die, centrifuging at 4000rpm for 20min, scraping off the redundant needle body solution, drying in a normal-temperature drying oven for 1h, adding the base layer solution, centrifuging at 4000rpm for 20min again, drying the microneedle female die in the normal-temperature drying oven for 24h at normal temperature, and demolding to obtain the b-type haemophilus influenzae conjugate vaccine soluble microneedle.

Table 3 formulation of different adjuvants

Example 4: activity and in vivo immunogenicity comparison of soluble microneedle patch containing different adjuvants for Haemophilus influenzae type b conjugate vaccine

(1) Activity detection of soluble microneedle of Haemophilus influenzae type b conjugate vaccine

After the soluble micro-needle of the haemophilus influenzae type b conjugate vaccine is prepared according to different prescriptions in the embodiment 3, the needle body of the micro-needle is dissolved in physiological saline, the activity of the vaccine is detected by adopting an immune double diffusion method, and the activity of the vaccine in the Hib conjugate vaccine in the micro-needle is detected by using the hyperimmune serum of the haemophilus influenzae type b and tetanus toxoid antibody. It is normal that both the Hib conjugate vaccine and the Haemophilus influenzae type b hyperimmune serum and tetanus toxoid antibody form a precipitate line. The results are shown in Table 4.

(2) Soluble microneedle immunogenicity detection for haemophilus influenzae type b conjugate vaccines

After the soluble microneedles of the haemophilus influenzae type b conjugate vaccine were prepared according to the different recipes of example 3, the immunogenicity of the conjugate vaccine was confirmed using a mouse as a model.

Selecting SPF-level mice, wherein each group comprises ten mice, removing body hair of all experimental groups and two sides of the back spine of a guinea pig through an electric hair remover, pressing the administration microneedle patch on the back skin of the mice for 3min by hands respectively for the rest groups, fixing the microneedle patch on the back skin by using sub-sensitive adhesive cloth, and stripping the patch and the pressure-sensitive adhesive after 4 hours. Subcutaneously injecting for 2 times on 1 and 14 days, and collecting blood on 21-28 days. HibIgG antibodies were assayed by the Elisa method, with a physiological sodium chloride solution control. Coating a 96-well enzyme label plate by using a coating buffer solution containing 2 mu g/ml of Hib PRP-tyramine, wherein the concentration of the enzyme label plate is 100 mu l/well, and the temperature is kept overnight at 4 ℃; incubating for 1h at 37 ℃ with PBS containing 20% calf serum; diluting the sample by multiple times, adding the diluted sample into an enzyme label plate, incubating for 1h at the temperature of 37 ℃ at a concentration of 100 mu l/hole; the standard product contains 20 mu g of anti-HibPRP antibody, a standard curve is drawn by using the anti-HibPRP antibody, and the content of 2 parts of quality control serum antibody is respectively 2.000 mu g/ml and 8.987 mu g/ml; adding alkaline phosphatase-labeled goat anti-mouse IgG, and incubating at 37 ℃ for 1 h; adding 100 mu l of substrate solution, and incubating for 1h at 37 ℃; the reaction was stopped with 50. mu.l of stop solution, the A405 values were read by a microplate reader and the serum was analyzed for the proportion of specific IgG1 and IgG2a antibodies to Hib, the results of which are shown in Table 4.

Table 4 comparison of the activity and in vivo immunogenicity of microneedle vaccines (mean% ± SD%, n ═ 10)

Note △ P <0.05 compared to blank and P <0.05 compared to positive control;

and (4) conclusion:

1) compared with the blank group, the experimental groups numbered 3.4, 3.5, 3.6, 3.7, 3.8 and 3.9 can obviously increase the immune activity of the microneedle vaccine (P <0.05), while the other experimental groups have no obvious difference (P >0.05) from the blank group and have no statistical significance;

2) compared with a positive control group, the experimental groups numbered 3.4, 3.6, 3.7, 3.8 and 3.9 can obviously increase the immunological activity of the microneedle vaccine, have obvious difference (P <0.05), and have no statistical significance when the other experimental groups are not obviously different from the positive control group (P > 0.05).

3) The adjuvant (numbered 3.6 and 3.7 experimental groups) formed by combining the SPO1, the astragalus polysaccharide and the 3-de-O-acylated monophosphoryl lipid A is adopted, the prepared soluble micro-needle patch for the haemophilus influenzae type b conjugate vaccine has high HibIgG antibody level and good immune effect, and the adjuvant effect of the combination of the three components is better than that of the combination of the two or single use of the three components; and the content ratio of the adjuvant to the vaccine is (1: 1) - (2: 1).

4) The number of the experimental group is 3.6, and the optimal formula is that the optimal content ratio of the SPO1, the astragalus polysaccharide and the 3-de-O-acylated monophosphoryl lipid A is 1: 1: 2, the optimal content ratio of adjuvant and vaccine is 1: 1.

5) the experimental group numbered 3.6, with a significantly higher ratio of IgG2a/IgG1 than the other groups and closest to 1, indicates the best effect of balancing the immune responses of Th1/Th2 types; furthermore, the best immunization effect was seen from the values of IgG antibodies.

Claims (7)

1. A quick-release b-type haemophilus influenzae conjugate vaccine soluble microneedle patch comprises a needle body and a basal layer, and is characterized in that the needle body consists of b-type haemophilus influenzae conjugate vaccine, adjuvant and water-soluble high polymer material, and the basal layer is made of flexible high polymer material; the content ratio of the adjuvant to the Haemophilus influenzae type b conjugate vaccine is (1: 1) - (2: 1), and the adjuvant is one or more of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A.

2. The immediate-release Haemophilus influenzae type b conjugate vaccine soluble microneedle patch as claimed in claim 1, wherein the water-soluble polymer material is a mixture of PVP and Gantrez, and the content ratio of PVP to Gantrez is 3: 1.

3. The immediate-release soluble microneedle patch for the haemophilus influenzae type b conjugate vaccine according to claim 1, wherein the adjuvant is a mixture of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid a, and the content ratio of the three is SPO 1: astragalus polysaccharide: 3-de-O-acylated monophosphoryl lipid a ═ (1: 1: 2) - (1: 2: 1).

4. The immediate-release Haemophilus influenzae type b conjugate vaccine dissolvable microneedle patch according to claim 1, wherein said needle body further comprises a heat-resistant protective agent.

5. The immediate-release Haemophilus influenzae type b conjugate vaccine soluble microneedle patch according to claim 4, wherein the heat-resistant protective agent is trehalose, and the flexible high polymer material is hypromellose.

6. The immediate-release Haemophilus influenzae type b conjugate vaccine soluble microneedle patch according to any one of claims 1 to 5, wherein the needle body is composed of a Haemophilus influenzae type b conjugate vaccine, an adjuvant, a water-soluble polymer material and trehalose, wherein the content ratio of the adjuvant to the Haemophilus influenzae type b conjugate vaccine is 1: 1, adjuvant is 1: 2: 1 of SPO1, astragalus polysaccharide and 3-de-O-acylated monophosphoryl lipid A, and the water-soluble high molecular material is the mixture of PVP and Gantrez with the content ratio of 3: 1; the basal layer is hydroxypropyl methylcellulose.

7. The method for preparing the immediate release haemophilus influenzae type b conjugate vaccine dissolvable microneedle patch of claim 1, comprising the steps of:

1) preparing a needle body solution: dissolving the water-soluble high polymer material, the haemophilus influenzae type b conjugate vaccine and the adjuvant with water according to the prescription amount, and uniformly mixing to obtain a needle body matrix solution;

2) preparation of base layer solution: dissolving a prescription amount of flexible high polymer material by using water to obtain a substrate solution;

3) preparation of soluble microneedles: injecting the needle body solution into the microneedle female die, centrifuging at 4000rpm for 20min, scraping off the redundant needle body solution, drying in a normal-temperature drying oven for 1h, adding the base layer solution, centrifuging at 4000rpm for 20min again, drying the microneedle female die at normal temperature for 24h, and demolding to obtain the microneedle female die.

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