CN110680911A - Japanese encephalitis vaccine soluble microneedle patch and preparation method thereof - Google Patents
Japanese encephalitis vaccine soluble microneedle patch and preparation method thereof Download PDFInfo
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- CN110680911A CN110680911A CN201810718919.2A CN201810718919A CN110680911A CN 110680911 A CN110680911 A CN 110680911A CN 201810718919 A CN201810718919 A CN 201810718919A CN 110680911 A CN110680911 A CN 110680911A
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- japanese encephalitis
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- 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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Abstract
The invention provides a Japanese encephalitis vaccine soluble microneedle patch which comprises a needle body and a back lining, wherein the needle body consists of a Japanese encephalitis vaccine, a matrix material and an adjuvant, the adjuvant is a mixture of MDP and GM-CSF, and the content ratio of the adjuvant to the Japanese encephalitis vaccine is (1:1) - (1: 3). The Japanese encephalitis vaccine soluble microneedle prepared by the invention has good immune effect and high mechanical strength of the needle body, is expected to replace Japanese encephalitis vaccine injection, and realizes painless minimally invasive administration.
Description
Technical Field
The invention relates to the technical field of microneedle administration of vaccines, and provides a soluble microneedle patch for a Japanese encephalitis vaccine and a preparation method thereof.
Background
Epidemic encephalitis B is an acute infectious disease affecting the central nervous system caused by encephalitis B virus, often causes death of patients or leaves nervous system sequelae, and encephalitis B vaccine is an effective measure for preventing epidemic encephalitis B. The existing Japanese encephalitis vaccine is generally administered through needle injection, and the compliance of patients is poor, so a novel vaccine administration route is urgently needed. In addition, the protein drugs are easily decomposed by the proteases in the gastrointestinal tract or the first-pass effect of the liver by oral administration, resulting in reduced activity, thereby affecting the bioavailability. Transdermal Drug Delivery System (TDDS) is a new technology of drug delivery which is popular in recent years, which can avoid the problems of poor patient compliance caused by the above-mentioned needle injection and the problems of oral administration of the first pass effect of the digestive tract and liver, and skin is favored as the largest immune organ of human body. However, transdermal delivery of water-soluble small molecule as well as macromolecular drugs has been problematic due to the presence of stratum corneum barriers.
Various physical permeation-promoting techniques are currently used to promote transdermal delivery of drugs, such as microneedles, iontophoresis, electrothermal porogenesis, ultrasound permeation promotion, and magnetic field introduction. Compared with the prior art, other physical permeation-promoting technologies can hardly control the administration position and dosage, and the microneedle can control the administration position by changing the length of the microneedle, and for a soluble microneedle, the microneedle can accurately control the administration dosage by controlling the drug content of the tip of the cast microneedle, so that the microneedle administration has unique advantages in both aspects, and the research in the field of microneedles raises the temperature year by year. The micro needles (microneedles) are an array combination with a plurality of micro needles, can penetrate into the stratum corneum of the skin to enter the epidermis layer without touching the nerve endings of the dermis layer, achieve painless minimally invasive administration, and the administration effect breaks through the traditional transdermal administration preparation. Compared with other types of microneedles, the soluble microneedle has the advantages of large drug loading, capability of being automatically degraded after penetrating into the skin without causing harmful residues, capability of achieving sustained and controlled release by screening different matrix material proportions and the like, and becomes the most studied microneedle type at present.
There is no example of loading Japanese encephalitis vaccine into soluble microneedle in the current domestic and foreign patents and literature. Similarly, patent publication No. IN2008CN02444A discloses that the administration of encephalitis vaccine into coated microneedles is well known to be very different from soluble microneedles IN terms of manufacturing process and drug loading, so it is difficult to use (Indermun S, Luttge R, Choonara Y E, et al. current enhancements IN the manufacture of the fabrics for transdermal delivery [ J ]. Journal of Controlled Release 2014,185(185): 130-.
The adjuvant is a non-specific immunopotentiator which can enhance the immune response of the organism to antigens or change the type of immune response when injected with antigens or injected into the organism in advance, the action mechanism of the immunologic adjuvant can be divided into ① antigen sustained release (antigen repository effect) ② upregulating various cytokines and chemokines, ③ recruiting immune cells to the injection site, ④ enhancing the uptake and presentation of antigens, ⑤ activating Antigen Presenting Cells (APCs), promoting the mature antigen presentation to be transported to draining lymph nodes, ⑥ activating inflammatory bodies, and the like, the immune enhancement effect of different adjuvants or adjuvants is different, therefore, aiming at different vaccines, the influence of different adjuvants and immune pathways on the immune protection effect of Eimeria tenella SO7 antigen can be achieved (J. Proc., 2007, 6929 (9):697, Pengjinpai Bighua, etc.: veterinary No. Shisanjie et al J. Immunity adjuvant, No. Shisanjie et al J. Shisanjie et al., Shisanjie et al.: No. Shi. Shisanjie et al.: 1, No. Shi. Shisanjie et al).
In addition to the advantages mentioned above, dissolvable microneedles suffer from certain limitations, such as possibly insufficient mechanical strength relative to other types of microneedles. According to the reference (Park J H, Allen M G, Prausnitz MR. Polymer microelectronics for controlled-release drug delivery [ J ]. pharmaceutical research,2006,23(5): 1008-. Therefore, screening of suitable microneedle drug loading ratio has a certain meaning for improving the mechanical strength of soluble microneedles.
In summary, how to develop a soluble microneedle with a suitable prescription, a good immune effect and a good mechanical strength for a Japanese encephalitis vaccine is a technical problem which needs to be solved urgently by those skilled in the art.
Disclosure of Invention
The invention aims to provide a Japanese encephalitis vaccine soluble microneedle patch and a preparation method thereof, and solves the bottleneck existing in the prior art.
The technical scheme adopted by the invention is as follows:
the needle body consists of a Japanese encephalitis vaccine, a matrix material and an adjuvant, wherein the adjuvant is a mixture of Muramyl Dipeptide (MDP) and human granulocyte-macrophage colony stimulating factor (GM-CSF), and the content ratio of the adjuvant to the Japanese encephalitis vaccine is (1:1) - (1: 3).
Preferably, the matrix material is Hyaluronic Acid (HA).
Preferably, the content ratio of GM-CSF to MDP in the adjuvant is (3:1) - (1: 3).
More preferably, the content ratio of GM-CSF to MDP in the adjuvant is 1: 2.
preferably, the content ratio of the adjuvant to the Japanese encephalitis vaccine is 1: 2.
preferably, the matrix material accounts for 30-50% of the needle body by mass.
Preferably, the needle body comprises the following components in percentage by weight: the weight percentage of the Japanese encephalitis vaccine is 40%, the weight percentage of GM-CSF is 6.7%, the weight percentage of MDP is 13.3%, and the weight percentage of HA is 40%.
The preparation method of the Japanese encephalitis vaccine soluble microneedle patch comprises the following steps:
(1) negative mold preparation of microneedles
Preparing a polydimethoxysiloxane female die required by the Japanese encephalitis vaccine soluble microneedle by adopting a reverse die method;
(2) first-time centrifugal mold-entering
Mixing and dissolving the components according to the prescription amount into uniform needle body liquid, coating a proper amount of the needle body liquid on a female die manufactured by an inverse die method, putting the female die into a 96-hole plate centrifuge, and centrifuging to enable the needle body liquid to enter holes of the female die; then, taking out the female die and scraping off the solution outside the hole;
(3) second time centrifugal mold
Dissolving the backing into uniform backing liquid, coating a proper amount of the backing liquid on the female die obtained in the step (1), putting the female die into a 96-hole plate centrifuge, and centrifuging to enable the backing liquid to enter the holes of the female die; and then putting the mixture into an oven, drying the mixture at 37 ℃, taking out the dried mixture, and demolding to obtain the product.
Compared with the prior art, the soluble micro-needle patch for the Japanese encephalitis vaccine and the preparation method thereof provided by the invention have the following beneficial effects:
① the invention selects the compound immunologic adjuvant with specific content ratio to combine with the Japanese encephalitis vaccine, so that the Japanese encephalitis vaccine soluble microneedle has the best immune enhancement effect.
② the invention screens proper matrix material mass ratio, so that the Japanese encephalitis vaccine soluble microneedle has moderate mechanical property, and the drug delivery success rate of the microneedle is improved.
③ the soluble microneedle prepared by the invention is a layered microneedle, which can make the dosage of the microneedle easier to control and can not cause drug waste.
In a word, the Japanese encephalitis vaccine soluble microneedle prepared by the invention has good immune effect and high mechanical strength of the needle body, is expected to replace Japanese encephalitis vaccine injection, and realizes painless minimally invasive administration.
Drawings
Fig. 1 is a schematic flow chart of preparing a microneedle negative mold;
FIG. 2 is a schematic flow chart of the process for preparing the Japanese encephalitis vaccine microneedles according to 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 negative mold preparation of Japanese encephalitis vaccine microneedles
Referring to fig. 1, the invention adopts a reverse mold method to prepare a female mold of a Japanese encephalitis vaccine microneedle, and comprises the following steps:
and (3) placing the metal male mold microneedle in a cuboid container, wherein the needle point is upward. Preparing Polydimethoxysiloxane (PDMS) and a curing agent according to a mass ratio of 10:1, and pouring into a rectangular container; placing the container in a vacuum drying oven, and respectively setting the parameters of vacuum degree of 0.07MPa and time of 5min to remove bubbles in the mixed solution; and then putting the container into an oven, setting the temperature parameter at 50 ℃, taking out the container after 5-8 h, and demolding to obtain the PDMS microneedle female mold.
Example 2 preparation of Japanese encephalitis vaccine microneedles
Referring to fig. 2, the method for preparing the Japanese encephalitis vaccine microneedle by using the secondary centrifugation injection molding method comprises the following steps:
1) mixing the components in the prescription in proportion, dissolving with appropriate amount of solvent (deionized water) to obtain uniform mixed solution (needle body fluid), placing appropriate amount of needle body fluid into a centrifuge tube, placing in a centrifugal precipitator, centrifuging to remove bubbles in the needle body fluid, and standing for use.
2) Pouring the needle body fluid obtained after centrifugation in the step 1) on a PDMS mold prepared in the embodiment 1 of the invention, and then placing the PDMS mold in a desk type low-speed centrifuge, and centrifuging at a rotating speed of 3000rpm for 5min to inject the needle body fluid into the holes of the mold; after centrifugation, taking out the mold, and scraping off the solution on the surface of the mold to only reserve the solution in the holes;
3) dissolving the composite material without the medicine backing layer into uniform liquid (namely backing liquid) by using a solvent (deionized water), putting a proper amount of the backing liquid into a centrifuge tube, putting the centrifuge tube into a centrifugal precipitator, centrifuging to remove air bubbles in the backing liquid, and standing for later use.
4) Pouring the backing solution obtained after centrifugation in the step 3) on the PDMS mold obtained in the step 2), and then placing the PDMS mold in a desk type low-speed centrifuge, and centrifuging at the rotating speed of 3000rpm for 3min to inject the backing solution into the holes of the mold; and (4) after centrifugation, taking out the mold, putting the mold into an oven, drying the mold at 37 ℃ for 8 hours, taking out the mold, and demolding to obtain the finished product of the Japanese encephalitis vaccine microneedle patch.
Example 3 Effect of the use of different immunological adjuvants on the Immunity Effect of the soluble microneedles of Japanese encephalitis vaccine
Assuming that the ratio of the immunoadjuvant to the total mass of the needle body is a constant value, only the components of the immunoadjuvant are changed, and the influence of different combinations of immunoadjuvants on the immunization effect of the microneedles as shown in table 1 is examined, the microneedles and the female mold according to the present embodiment are prepared by referring to example 1 and example 2 of the present invention.
TABLE 1 Effect of different immunological adjuvants on the immunization effect of soluble microneedles of Japanese encephalitis vaccine
After preparing soluble microneedles for 11 microneedles in table 1 by the methods of examples 1 and 2 of the present invention, the immune effect of the soluble microneedles was tested as follows:
respectively pricking 18 soluble microneedles into the back skin of 18 unhaired rats by using an administration device, taking out after the microneedles are completely dissolved, and carrying out secondary immunization on the rats by the same method after a week interval. Serum was taken from rat veins seven days after the second booster immunization, and the Japanese encephalitis vaccine-specific IgG level in the serum was quantified using an ELISA (enzyme-linked immunosorbent assay) kit.
The results of the tests are shown in Table 2.
TABLE 2 Effect of combinations of different immunological adjuvants on the immunization effect of soluble microneedles of Japanese encephalitis vaccine
As can be seen from table 2, the immune effect is completely different when the immune adjuvant is added compared with that when the immune adjuvant is not added, and the immune enhancement effect of different adjuvants on the soluble microneedles of the Japanese encephalitis vaccine is different, when a single adjuvant is used for administration, the immune enhancement effect of MDP is the best, and then MPL, GM-CSF and aluminum hydroxide gel are used; the compound adjuvant has better immune enhancement effect than that of a single adjuvant, wherein the serum antibody level corresponding to the microneedle with the prescription No. 6 is the highest, which shows that the immune synergy of the Japanese encephalitis vaccine is most obvious when the MDP adjuvant and the GM-CSF adjuvant are used in combination, and therefore, the immune adjuvant selects the mixture of the MDP adjuvant and the GM-CSF adjuvant.
Example 4 Effect of the content of the Complex immunologic adjuvant on the Immunity Effect of the soluble microneedles of the Japanese encephalitis vaccine
On the basis of example 3, assuming that the ratio of the composite immunoadjuvant to the total mass of the needle body is a constant value, and the components of the composite immunoadjuvant are fixed, only the content change of each component in the immunoadjuvant is changed, and the influence of the composite immunoadjuvant with different ratios on the microneedle immune effect as shown in table 3 is examined.
Similarly, the microneedles and the female mold according to the present embodiment are prepared according to the methods of example 1 and example 2 of the present invention. The immune effect of each soluble microneedle formulation in table 3 was tested and the test procedure was according to example 3 of the present invention.
TABLE 3 Effect of different contents of composite immunoadjuvants on microneedle immunization
The results are shown in Table 4.
TABLE 4 Effect of different amounts of adjuvant combinations on the immunization of microneedles
As can be seen from the combination of tables 3 and 4,
1) the immunopotentiation effect is more obvious along with the increase of the MDP content in the compound adjuvant, but when the ratio of GM-CSF: when MDP reaches 1:3 (namely prescription 5), the immune enhancement effect is weakened.
2) When GM-CSF: when MDP is 1:2, the immunopotentiation effect is the best, so it is the best prescription.
Example 5 Effect of different ratios of composite adjuvant to vaccine on the Immunity of microneedles
Based on example 4, assuming that the ratio of GM-CSF to MDP in the adjuvant composition is constant (1:2), the ratio of the matrix material is fixed, and the ratio of the adjuvant composition to the vaccine is changed only, the effect of different ratios of adjuvant composition and vaccine on the immunization effect of the microneedles as shown in table 5 was examined.
Similarly, the microneedles and the female mold according to the present embodiment are prepared according to the methods of example 1 and example 2 of the present invention. The immune effect of each soluble microneedle formulation in table 5 was tested and the test procedure was according to example 3 of the present invention.
TABLE 5 Effect of different proportions of the composite immunologic adjuvant and the vaccine amount on the immunization effect of the microneedle
| No | Composite immunologic adjuvant and Japanese encephalitis vaccine | Matrix material HA (wt%) |
| 1 | And (3) compound immunologic adjuvant: encephalitis B vaccine 1:3 | 40 |
| 2 | And (3) compound immunologic adjuvant: encephalitis B vaccine 1:2 | 40 |
| 3 | And (3) compound immunologic adjuvant: encephalitis B vaccine 1:1 | 40 |
The results are shown in Table 6.
TABLE 6 influence of different proportions of composite immunologic adjuvant and vaccine amount on microneedle immunization effect
As shown in table 6, as the ratio of the encephalitis B vaccine to the composite immunologic adjuvant is increased from (1:1) to (2:1), namely, the proportion of the encephalitis B vaccine is increased, the immune enhancement effect is more remarkable; when the ratio is increased to (3:1), the immunopotentiation effect is reduced with a decrease in the amount of the combined immunoadjuvant, even if the vaccine ratio continues to increase. Therefore, when the ratio of the Japanese encephalitis vaccine to the composite immunologic adjuvant is 2:1 (namely, the prescription 2), the immune enhancement effect is optimal, so that the Japanese encephalitis vaccine is used as the optimal prescription.
Example 6 Effect of different matrix Material occupancy ratios on microneedle Performance
According to the literature (Park J H, Allen M G, Prausnitz M r. polymer semiconductors for controlled-release drug delivery [ J ]. Pharm Res,2006,23(5): 1008-19), the mechanical properties of microneedles decrease as the amount of drug loaded in the microneedles increases. Therefore, in addition to the evaluation of the immune effect, the appropriate ratio of the matrix material of the microneedle is determined by combining the mechanical properties of the microneedle, and the content of the matrix material is changed only assuming that the mass ratio of the composite adjuvant to the vaccine is a certain value, and the investigation formula is shown in table 7.
Mechanical property test of soluble microneedles: after the microneedles were inserted into the skin of the isolated rat with a force of 5N using an applicator, the area where the skin was inserted was stained with methylene blue, and the ratio of the number of blue dots to the number of microneedles was calculated to make 6 groups parallel.
Similarly, the microneedles and the female mold according to the present embodiment are prepared according to the methods of example 1 and example 2 of the present invention. The immune effect of each soluble microneedle formulation in table 7 was tested and the test procedure was according to example 3 of the present invention.
Table 7 influence of different matrix material occupancy on mechanical properties and immune effect of microneedles
As shown in table 7, the mechanical strength of the microneedles increased with the increase in the ratio of the matrix material, and the number of needles that can be inserted into the skin for administration increased, but the drug loading of the microneedles decreased. When the proportion of the matrix material HA is 40% (namely, formula 2), the mechanical strength is medium, the immune effect is good, and the HA is used as the optimal formula.
In conclusion, the optimal prescription of the Japanese encephalitis vaccine soluble microneedle is as follows: 40 wt% Japanese encephalitis vaccine, 6.7 wt% GM-CSF, 13.3 wt% MDP, 40 wt% HA.
Example 7 Japanese encephalitis vaccine soluble microneedle skin puncture experiment
After soluble microneedles were prepared according to the methods of examples 1 and 2 above and the optimal formulation screened in the previous examples (40 wt% Japanese encephalitis vaccine, 6.7 wt% GM-CSF, 13.3 wt% MDP, 40 wt% HA), they were pressed into the skin of the isolated mouse with a 5N force using a dispenser for 10s, fixed in 10% formaldehyde solution and left to be histologically sectioned. The observation of the slicing result shows that the microneedle punctures the cuticle and enters the epidermis, which shows that the soluble microneedle prepared by the invention can puncture the skin effectively.
Claims (8)
1. The soluble micro-needle patch for the Japanese encephalitis vaccine comprises a needle body and a back lining, and is characterized in that the needle body consists of the Japanese encephalitis vaccine, a matrix material and an adjuvant, wherein the adjuvant is a mixture of MDP and GM-CSF, and the content ratio of the adjuvant to the Japanese encephalitis vaccine is (1:1) - (1: 3).
2. The Japanese encephalitis vaccine soluble microneedle patch according to claim 1, wherein the matrix material is hyaluronic acid.
3. The Japanese encephalitis vaccine soluble microneedle patch according to claim 1, wherein the content ratio of GM-CSF to MDP in the adjuvant is (3:1) - (1: 3).
4. The Japanese encephalitis vaccine soluble microneedle patch according to claim 3, wherein the content ratio of GM-CSF to MDP in the adjuvant is 1: 2.
5. the Japanese encephalitis vaccine soluble microneedle patch according to claim 1, wherein the content ratio of adjuvant to Japanese encephalitis vaccine is 1: 2.
6. the Japanese encephalitis vaccine soluble microneedle patch according to claim 1, wherein the matrix material accounts for 30-50% of the needle body by mass.
7. The Japanese encephalitis vaccine soluble microneedle patch according to any one of claims 1 to 6, wherein the needle body contains the following components: the weight percentage of the Japanese encephalitis vaccine is 40 percent, the weight percentage of GM-CSF is 6.7 percent, the weight percentage of MDP is 13.3 percent, and the weight percentage of hyaluronic acid is 40 percent.
8. The method for preparing a Japanese encephalitis vaccine soluble microneedle patch according to claim 1, comprising the steps of:
(1) negative mold preparation of microneedles
Preparing a polydimethoxysiloxane female die required by the Japanese encephalitis vaccine soluble microneedle by adopting a reverse die method;
(2) first-time centrifugal mold-entering
Mixing and dissolving the components according to the prescription amount into uniform needle body liquid, coating a proper amount of the needle body liquid on a female die manufactured by an inverse die method, putting the female die into a 96-hole plate centrifuge, and centrifuging to enable the needle body liquid to enter holes of the female die; then, taking out the female die, and scraping the solution outside the holes;
(3) second time centrifugal mold
Dissolving the backing into uniform backing liquid, coating a proper amount of the backing liquid on the female die obtained in the step (1), putting the female die into a 96-hole plate centrifuge, and centrifuging to enable the backing liquid to enter the holes of the female die; and then putting the mixture into an oven, drying the mixture at 37 ℃, taking out the dried mixture, and demolding to obtain the product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112516449A (en) * | 2020-12-03 | 2021-03-19 | 昆明理工大学 | Soluble microneedle array and preparation method thereof |
| CN114150022A (en) * | 2021-12-06 | 2022-03-08 | 中国科学院精密测量科学与技术创新研究院 | Biochemical molecular cell delivery method based on plant micro-nano structure and application |
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| CN102793917A (en) * | 2012-07-24 | 2012-11-28 | 北京中联康生物科技有限公司 | Porcine encephalitis B vaccine and preparation method thereof |
| CN107157933A (en) * | 2017-05-04 | 2017-09-15 | 同济大学 | A kind of albumen self assembly novel nano vaccine 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102793917A (en) * | 2012-07-24 | 2012-11-28 | 北京中联康生物科技有限公司 | Porcine encephalitis B vaccine 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 |
| CN107157933A (en) * | 2017-05-04 | 2017-09-15 | 同济大学 | A kind of albumen self assembly novel nano vaccine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112516449A (en) * | 2020-12-03 | 2021-03-19 | 昆明理工大学 | Soluble microneedle array and preparation method thereof |
| CN112516449B (en) * | 2020-12-03 | 2023-02-21 | 昆明理工大学 | Soluble microneedle array and preparation method thereof |
| CN114150022A (en) * | 2021-12-06 | 2022-03-08 | 中国科学院精密测量科学与技术创新研究院 | Biochemical molecular cell delivery method based on plant micro-nano structure and application |
| CN114150022B (en) * | 2021-12-06 | 2023-08-22 | 中国科学院精密测量科学与技术创新研究院 | Biochemical molecular cell delivery method based on plant micro-nano structure and application |
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