阅读说明：本技术 一种速释b型流感嗜血杆菌结合疫苗可溶性微针贴及其制备方法 (Quick-release type-b haemophilus influenzae conjugate vaccine soluble microneedle patch and preparation method thereof ) 是由 张庶民 马凤森 周荔葆 廖辉 陈中秋 刘苗苗 吴铮 辛小韵 于 2018-07-04 设计创作，主要内容包括：本发明提供了一种速释b型流感嗜血杆菌结合疫苗可溶性微针贴,包括针体和基底层,所述的针体由b型流感嗜血杆菌结合疫苗、佐剂、水溶性高分子材料组成,所述的基底层为柔性高分子材料；其中,佐剂与b型流感嗜血杆菌结合疫苗的含量比为(1：1)～(2：1),所述的佐剂为SPO1、黄芪多糖、3-脱-O-酰化单磷酰脂质A中的一种或几种。本发明提供的b型流感嗜血杆菌结合疫苗可溶性微针贴,能够实现平衡Th1/Th2型的免疫应答,以及疫苗在皮肤组织内快速释放。(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.)

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.

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.