CN108379573B - Pertussis vaccine microneedle array and preparation method thereof - Google Patents
Pertussis vaccine microneedle array and preparation method thereof Download PDFInfo
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- CN108379573B CN108379573B CN201810254703.5A CN201810254703A CN108379573B CN 108379573 B CN108379573 B CN 108379573B CN 201810254703 A CN201810254703 A CN 201810254703A CN 108379573 B CN108379573 B CN 108379573B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/099—Bordetella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
Abstract
The invention discloses a preparation method of a pertussis vaccine microneedle array, which comprises the following steps: 1) using a microneedle array mould, wherein the microneedle array mould comprises a substrate, a rectangular groove is arranged on the upper surface of the substrate, and a conical groove with an array tip end downwards is arranged on the bottom wall of the rectangular groove; 2) uniformly mixing a pertussis toxin aqueous solution and an adjuvant aqueous solution to obtain a solution I, adding the solution I into a rectangular groove, standing under a vacuum condition to enable the solution I to fill the conical groove, sucking off the excess solution I outside the conical groove, and drying; 3) and (3) coating a high molecular polymer aqueous solution in the rectangular groove, drying, stripping the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum. The pertussis vaccine microneedle array obtained by the method is accurate in dosage, standard in product and safe to operate.
Description
Technical Field
The invention belongs to the technical field of vaccine administration equipment, and particularly relates to a pertussis vaccine microneedle array and a preparation method thereof.
Background
Pertussis is an acute respiratory infectious disease caused by Bordetella pertussis, generally caused by Bordetella pertussis (abbreviated as Bordetella pertussis), and also caused by Bordetella bronchiseptica and Bordetella parapertussis belonging to the same genus. Bordetella pertussis is a gram-negative bacterium that produces several pathogenic substances including pertussis toxin, tracheal cytotoxin, adenylate cyclase toxin, thermolabile toxin, endotoxin, and the like. Pertussis toxin can mobilize lymphocytes in patient's lymph tissue to peripheral blood and trachea, and cytotoxin can specifically damage tracheal ciliated epithelial cells to cause degeneration and necrosis. Pertussis patients, recessive infectors and carriers are the sources of infection. The infectivity is strongest from the end of latent period to 2-3 weeks after illness. Pertussis is transmitted by respiratory droplets, the susceptibility of children under 5 years old is the highest, and the pertussis infection rate is not different from that of non-vaccinated people after the children are vaccinated for 10 years.
Generally, the pertussis vaccine is administered by injection, and although it is directly effective, the target to be vaccinated is usually a young child, and the injection causes pain and skin injury, and is difficult to operate, cannot be used by itself, and usually causes many difficulties in vaccination.
Transdermal administration of microneedles is a new mode of administration that avoids the above disadvantages. The small needle with micron scale can penetrate epidermal layer to reach dermal layer and release medicine into body without contacting nervous system to complete medicine administration.
But the current method for preparing the microneedle transdermal drug delivery can not achieve accurate metering. There has also been no report combining the traditional pertussis vaccine with microneedle administration techniques.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a pertussis vaccine microneedle array.
A second object of the present invention is to provide a method for preparing a pertussis vaccine microneedle array.
The technical scheme of the invention is summarized as follows:
a preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) using a microneedle array mould, wherein the microneedle array mould comprises a substrate (1), a rectangular groove (2) is formed in the upper surface of the substrate (1), and a conical groove (3) with an array tip end downward is formed in the bottom wall of the rectangular groove (2);
2) uniformly mixing 0.1-1mg/ml pertussis toxin aqueous solution and 0.1-100mg/ml adjuvant aqueous solution according to the volume ratio of 1:1-10 to obtain a first solution, adding the first solution into the rectangular groove (2), standing for 10-60min under a vacuum condition to enable the first solution to fill the conical groove (3), sucking off the redundant first solution outside the conical groove (3), and drying for 30-120 min;
3) and (3) smearing a high-molecular polymer aqueous solution with the mass concentration of 10% -50% into the rectangular groove, drying for 8-12 hours, removing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum to obtain the pertussis vaccine microneedle array.
The adjuvant is preferably CpG.
The high molecular polymer is preferably one or more of polyethylene with molecular weight of 10000-.
The vacuum drying time is preferably 20 to 26 hours.
The pertussis vaccine microneedle array prepared by the method.
The invention has the advantages that:
according to the preparation method of the pertussis vaccine microneedle array, the microneedle array mold is used, and the rectangular grooves are formed, so that the liquid adding amount of each conical groove is the same when a solution containing a pertussis toxin aqueous solution is added into the rectangular grooves, the mold is kept still under a vacuum condition, the redundant solution outside the conical grooves (3) is sucked, the volume of each conical groove is fixed, and when the concentration of the pertussis toxin aqueous solution is fixed and the number of the conical grooves is fixed, the pertussis toxin dose of the whole microneedle array can be accurately obtained; in addition, the overflow of the liquid medicine of the pertussis toxin-containing aqueous solution without the rectangular groove can be avoided; in addition, the liquid level of the high molecular polymer aqueous solution in the rectangular groove (2) can be the same, and a product with uniform thickness after the high molecular polymer is dried is prepared. Through transdermal administration, the immune effect equivalent to or even better than that of injection is achieved. The pertussis vaccine microneedle array obtained by the method is accurate in dosage, standard in product and safe to operate.
Drawings
Fig. 1 is a schematic longitudinal section of a microneedle array mold for preparing a pertussis vaccine microneedle array.
Fig. 2 is a partially enlarged photograph of a pertussis vaccine microneedle array prepared by the method of the present invention.
Fig. 3 is a comparison of pertussis vaccine microneedle arrays of the present invention with subcutaneous immunization results.
Figure 4 is a comparison of the results of immunization with different doses of a pertussis vaccine microneedle array.
Detailed Description
The present invention is further illustrated by the following examples, which are provided to enable those skilled in the art to better understand the present invention and are not intended to limit the present invention in any way.
Pertussis Toxin (PTX).
Example 1
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) using a microneedle array mold, see fig. 1, the microneedle array mold comprises a substrate 1, wherein rectangular grooves 2 are arranged on the upper surface of the substrate 1, conical grooves 3 with 10 × 10 array tips downward are arranged on the bottom wall of the rectangular grooves 2, the volume of each conical groove 3 is 0.02 μ L, and the total volume of the conical grooves is 2 ul;
2) uniformly mixing 1mg/mL inactivated pertussis toxin aqueous solution and 10mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:1 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 15min under a vacuum condition to enable the first solution to fill the conical groove 3, absorbing the redundant first solution outside the conical groove 3, and drying for 60 min;
3) and (3) smearing a polyvinyl alcohol aqueous solution with the mass concentration of 30% viscosity average molecular weight of 20000 in the rectangular groove, drying for 10 hours, releasing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum for 24 hours to obtain the pertussis vaccine microneedle array, wherein a partial enlarged view of the microneedle array is shown in fig. 2.
Each pertussis vaccine microneedle array carries 1 μ g of pertussis toxin and 10 μ g of CpG adjuvant.
Experiment 1:
12 female Balb/c mice, 18-20g, were selected and randomly divided into two groups, 6 experimental groups, and were immunized intradermally with the pertussis vaccine microneedle array of example 1; control group 6 were immunized intradermally with injections containing 1 μ g pertussis toxin and 10 μ g of CpG adjuvant, and both groups were immunized once every three weeks and twice in total, and antibody levels were measured by blood sampling at three weeks and six weeks, respectively, as shown in fig. 3, and the experimental results show that: after the first immunization, the immune effect of the experimental group is obviously better than that of the control group; after the second immunization, the two immunizations have equivalent effects. The pertussis vaccine microneedle patch has similar immune effect to that of the traditional pertussis vaccine injection, and has the characteristic of single immunization and high efficiency.
Example 2
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) same as example 1, step 1);
2) uniformly mixing 0.1mg/mL inactivated pertussis toxin aqueous solution and 10mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:1 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 15min under a vacuum condition to enable the first solution to fill the conical groove 3, sucking off the redundant first solution outside the conical groove 3, and drying for 60 min;
3) same as example 1, step (3).
Each pertussis vaccine microneedle array carries a pertussis toxin amount of 0.1 μ g, and a CpG adjuvant amount of 10 μ g.
Example 3
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) same as example 1, step 1);
2) uniformly mixing 0.5mg/mL inactivated pertussis toxin aqueous solution and 10mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:1 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 15min under a vacuum condition to enable the first solution to fill the conical groove 3, sucking off the redundant first solution outside the conical groove 3, and drying for 60 min;
3) same as example 1, step (3).
Each pertussis vaccine microneedle array carries a pertussis toxin amount of 0.5 μ g, and a CpG adjuvant amount of 10 μ g.
Experiment 2:
24 female Balb/c mice, 18-20g, are randomly divided into 4 groups, 6 groups in example 1, and are immunized in skin by adopting the pertussis vaccine microneedle array in example 1; example 2 group 6 were immunized intradermally with pertussis vaccine microneedle arrays of example 2, respectively; example 3 groups 6 were immunized intradermally with pertussis vaccine microneedle arrays of example 2, respectively; a blank group of 6 mice was immunized intradermally with PBS injection at pH 7.4.
Groups 4 were immunized once every three weeks for two times, and blood was collected at three weeks and six weeks for detection of antibody levels, as shown in FIG. 4,
the experimental results show that after the first immunization, the immunization effects of the microneedles with different dosages in the three groups of the embodiment are not obviously different; after the second immunization, the low dose 0.1 microgram group (example 2 group) had a significant drop in the level of immunity, the medium and high dose groups had comparable levels of immunity, and the medium dose group was slightly higher than the high dose group. The pertussis vaccine microneedle has the best immune effect on mice when the dosage of the pertussis vaccine microneedle is 0.5 microgram.
Example 4
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) using a microneedle array mold, see fig. 1, the microneedle array mold comprises a substrate 1, wherein rectangular grooves 2 are arranged on the upper surface of the substrate 1, conical grooves 3 with 10 × 10 array tips downward are arranged on the bottom wall of the rectangular grooves 2, the volume of each conical groove 3 is 0.02 μ L, and the total volume of the conical grooves is 2 ul;
2) uniformly mixing 1mg/mL inactivated pertussis toxin aqueous solution and 0.1mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:10 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 15min under a vacuum condition to enable the first solution to fill the conical groove 3, sucking off the redundant first solution outside the conical groove 3, and drying for 60 min;
3) and (3) smearing a polyvinyl alcohol aqueous solution with the mass concentration of 30% viscosity average molecular weight of 20000 in the rectangular groove, drying for 10 hours, removing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum for 24 hours to obtain the pertussis vaccine microneedle array.
Each pertussis vaccine microneedle array carries pertussis toxin in an amount of 0.182 μ g, and CpG adjuvant in an amount of 0.182 μ g.
Example 5
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) using a microneedle array mold, see fig. 1, the microneedle array mold comprises a substrate 1, wherein rectangular grooves 2 are arranged on the upper surface of the substrate 1, conical grooves 3 with 10 × 10 array tips downward are arranged on the bottom wall of the rectangular grooves 2, the volume of each conical groove 3 is 0.02 μ L, and the total volume of the conical grooves is 2 ul;
2) uniformly mixing 1mg/mL inactivated pertussis toxin aqueous solution and 100mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:5 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 10min under a vacuum condition to enable the first solution to fill the conical groove 3, absorbing the redundant first solution outside the conical groove 3, and drying for 120 min;
3) and (3) smearing a polyvinyl alcohol aqueous solution with the mass concentration of 16000 viscosity average molecular weight of 50% in the rectangular groove, drying for 12 hours, removing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum for 26 hours to obtain the pertussis vaccine microneedle array.
Each pertussis vaccine microneedle array was loaded with pertussis toxin in an amount of 0.33 μ g, CpG adjuvant 166.67.
Example 6
A preparation method of a pertussis vaccine microneedle array comprises the following steps:
1) using a microneedle array mold, see fig. 1, the microneedle array mold comprises a substrate 1, wherein rectangular grooves 2 are arranged on the upper surface of the substrate 1, conical grooves 3 with 10 × 10 array tips downward are arranged on the bottom wall of the rectangular grooves 2, the volume of each conical groove 3 is 0.02 μ L, and the total volume of the conical grooves is 2 ul;
2) uniformly mixing 1mg/mL inactivated pertussis toxin aqueous solution and 10mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:1 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove 2, standing for 60min under a vacuum condition to enable the first solution to fill the conical groove 3, absorbing the redundant first solution outside the conical groove 3, and drying for 30 min;
3) and (3) smearing a polyvinyl alcohol aqueous solution with the mass concentration of 10% and the viscosity average molecular weight of 200000 into the rectangular groove, drying for 8 hours, releasing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum for 20 hours to obtain the pertussis vaccine microneedle array.
Each pertussis vaccine microneedle array carries 1 μ g of pertussis toxin and 10 μ g of CpG adjuvant.
Experiments have shown that pertussis vaccine microneedle arrays were obtained using polyethylene having a molecular weight of 10000, polyethylene having a molecular weight of 100000, polypropylene having a molecular weight of 80000, polypropylene having a molecular weight of 150000, polymethyl acrylate having a molecular weight of 10000, polymethyl acrylate having a molecular weight of 50000, polymethyl methacrylate having a molecular weight of 80000, or polymethyl methacrylate having a molecular weight of 200000 instead of the polyvinyl alcohol having a molecular weight of 20000 in example 1, respectively, in the same manner as in example 1.
Claims (2)
1. A preparation method of a pertussis vaccine microneedle array is characterized by comprising the following steps:
1) using a microneedle array mould, wherein the microneedle array mould comprises a substrate (1), rectangular grooves (2) are formed in the upper surface of the substrate (1), 10 multiplied by 10 conical grooves (3) with downward array tips are formed in the bottom wall of each rectangular groove (2), the volume of each conical groove (3) is 0.02 mu L, and the total volume of each conical groove is 2 ul;
2) uniformly mixing 0.5mg/mL inactivated pertussis toxin aqueous solution and 10mg/mL CpG adjuvant aqueous solution according to the volume ratio of 1:1 to obtain a first solution, absorbing 100 microliters of the first solution, adding the first solution into the rectangular groove (2), standing for 15min under a vacuum condition to enable the first solution to fill the conical groove (3), sucking off the redundant first solution outside the conical groove (3), and drying for 60 min;
3) and (3) smearing a polyvinyl alcohol aqueous solution with the mass concentration of 30% viscosity average molecular weight of 20000 in the rectangular groove, drying for 10 hours, removing the membrane with the conical protrusions from the microneedle array mold, and drying in vacuum for 24 hours to obtain the pertussis vaccine microneedle array.
2. A pertussis vaccine microneedle array prepared by the method of claim 1.
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| WO2011150144A2 (en) * | 2010-05-28 | 2011-12-01 | 3M Innovative Properties Company | Aqueous formulations for coating microneedle arrays |
| CN104027324B (en) * | 2013-03-06 | 2017-12-15 | 中国科学院理化技术研究所 | A kind of soluble micropin vaccine paster and preparation method thereof |
| CA2981974C (en) * | 2014-04-24 | 2023-06-20 | Georgia Tech Research Corporation | Microneedles and methods of manufacture thereof |
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| CN104706626B (en) * | 2015-03-25 | 2018-02-23 | 北京化工大学 | A kind of microneedle patch for being easy to animal vaccine to be administered and preparation method thereof |
| CN107412943A (en) * | 2017-04-17 | 2017-12-01 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of soluble microneedle patch and preparation method thereof |
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