CN116807520A - Method for minimally invasive extraction of tissue fluid without damage and pain - Google Patents
Method for minimally invasive extraction of tissue fluid without damage and pain Download PDFInfo
- Publication number
- CN116807520A CN116807520A CN202310777701.5A CN202310777701A CN116807520A CN 116807520 A CN116807520 A CN 116807520A CN 202310777701 A CN202310777701 A CN 202310777701A CN 116807520 A CN116807520 A CN 116807520A
- Authority
- CN
- China
- Prior art keywords
- microneedle
- nitrate
- gel
- chloride
- needle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000605 extraction Methods 0.000 title claims abstract description 49
- 239000012530 fluid Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000006378 damage Effects 0.000 title claims abstract description 8
- 238000006731 degradation reaction Methods 0.000 claims abstract description 52
- 230000015556 catabolic process Effects 0.000 claims abstract description 50
- 239000000463 material Substances 0.000 claims abstract description 30
- 238000004132 cross linking Methods 0.000 claims abstract description 28
- 229920000642 polymer Polymers 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 238000010382 chemical cross-linking Methods 0.000 claims abstract description 5
- 239000007787 solid Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 92
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 82
- 239000011780 sodium chloride Substances 0.000 claims description 41
- 229910021538 borax Inorganic materials 0.000 claims description 18
- 239000004328 sodium tetraborate Substances 0.000 claims description 18
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 18
- 108010010803 Gelatin Proteins 0.000 claims description 14
- 239000008273 gelatin Substances 0.000 claims description 14
- 229920000159 gelatin Polymers 0.000 claims description 14
- 235000019322 gelatine Nutrition 0.000 claims description 14
- 235000011852 gelatine desserts Nutrition 0.000 claims description 14
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- 239000001110 calcium chloride Substances 0.000 claims description 9
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 9
- 239000000661 sodium alginate Substances 0.000 claims description 9
- 235000010413 sodium alginate Nutrition 0.000 claims description 9
- 229940005550 sodium alginate Drugs 0.000 claims description 9
- 229920001661 Chitosan Polymers 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 claims description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 6
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 5
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 5
- 229920002307 Dextran Polymers 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- IWOUKMZUPDVPGQ-UHFFFAOYSA-N barium nitrate Chemical compound [Ba+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O IWOUKMZUPDVPGQ-UHFFFAOYSA-N 0.000 claims description 4
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 4
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 4
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- VCJMYUPGQJHHFU-UHFFFAOYSA-N iron(3+);trinitrate Chemical compound [Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O VCJMYUPGQJHHFU-UHFFFAOYSA-N 0.000 claims description 4
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 4
- HSZCZNFXUDYRKD-UHFFFAOYSA-M lithium iodide Chemical compound [Li+].[I-] HSZCZNFXUDYRKD-UHFFFAOYSA-M 0.000 claims description 4
- IIPYXGDZVMZOAP-UHFFFAOYSA-N lithium nitrate Chemical compound [Li+].[O-][N+]([O-])=O IIPYXGDZVMZOAP-UHFFFAOYSA-N 0.000 claims description 4
- YIXJRHPUWRPCBB-UHFFFAOYSA-N magnesium nitrate Chemical compound [Mg+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O YIXJRHPUWRPCBB-UHFFFAOYSA-N 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 4
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 4
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 3
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 3
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 3
- 125000002091 cationic group Chemical group 0.000 claims description 3
- 229920002674 hyaluronan Polymers 0.000 claims description 3
- 229960003160 hyaluronic acid Drugs 0.000 claims description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 3
- INHCSSUBVCNVSK-UHFFFAOYSA-L lithium sulfate Inorganic materials [Li+].[Li+].[O-]S([O-])(=O)=O INHCSSUBVCNVSK-UHFFFAOYSA-L 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- RBTVSNLYYIMMKS-UHFFFAOYSA-N tert-butyl 3-aminoazetidine-1-carboxylate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)N1CC(N)C1 RBTVSNLYYIMMKS-UHFFFAOYSA-N 0.000 claims description 3
- BNGXYYYYKUGPPF-UHFFFAOYSA-M (3-methylphenyl)methyl-triphenylphosphanium;chloride Chemical compound [Cl-].CC1=CC=CC(C[P+](C=2C=CC=CC=2)(C=2C=CC=CC=2)C=2C=CC=CC=2)=C1 BNGXYYYYKUGPPF-UHFFFAOYSA-M 0.000 claims description 2
- JDXXTKLHHZMVIO-UHFFFAOYSA-N 2-(3-hydroxypropyl)guanidine Chemical compound NC(=N)NCCCO JDXXTKLHHZMVIO-UHFFFAOYSA-N 0.000 claims description 2
- FLCAEMBIQVZWIF-UHFFFAOYSA-N 6-(dimethylamino)-2-methylhex-2-enamide Chemical compound CN(C)CCCC=C(C)C(N)=O FLCAEMBIQVZWIF-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 2
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 2
- 239000004696 Poly ether ether ketone Substances 0.000 claims description 2
- 229920000691 Poly[bis(2-chloroethyl) ether-alt-1,3-bis[3-(dimethylamino)propyl]urea] Polymers 0.000 claims description 2
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- 229920002873 Polyethylenimine Polymers 0.000 claims description 2
- 239000004721 Polyphenylene oxide Substances 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims description 2
- UCXOJWUKTTTYFB-UHFFFAOYSA-N antimony;heptahydrate Chemical compound O.O.O.O.O.O.O.[Sb].[Sb] UCXOJWUKTTTYFB-UHFFFAOYSA-N 0.000 claims description 2
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims description 2
- 229910001626 barium chloride Inorganic materials 0.000 claims description 2
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 claims description 2
- 229910001863 barium hydroxide Inorganic materials 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- XEVRDFDBXJMZFG-UHFFFAOYSA-N carbonyl dihydrazine Chemical compound NNC(=O)NN XEVRDFDBXJMZFG-UHFFFAOYSA-N 0.000 claims description 2
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
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- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 2
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- XGZVUEUWXADBQD-UHFFFAOYSA-L lithium carbonate Chemical compound [Li+].[Li+].[O-]C([O-])=O XGZVUEUWXADBQD-UHFFFAOYSA-L 0.000 claims description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of biological medicine, and particularly relates to a method for extracting tissue fluid in a nondestructive painless minimally invasive manner. The method is based on a microneedle system, and the microneedle structure involved in the method comprises a substrate and a needle body. The substrate is of a patch-like structure, the needle body is of a solid conical or pyramid-like structure, the diameter of a circle circumscribing the bottom surface of the needle point is 50-800 mu m, the height is 100-2000 mu m, and the center distance between adjacent micro-needles is 50-1000 mu m. The main materials of the substrate and the needle body are prepared from natural polysaccharide and protein macromolecules with high biocompatibility, the polymers involved in the materials are mainly subjected to reversible physical crosslinking, and chemical crosslinking is assisted, so that the mechanical strength of the microneedle gel is ensured, and cyclic reversible gel forming and degradation can be realized. Adverse effects and even damage to components of the extracting solution caused by degrading the microneedles in a traditional enzymology mode are avoided, detection errors caused by the adverse effects and even damage are avoided, and the problem that the traditional microneedle gel tissue fluid extraction cannot be used for multiple times is solved.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a method for extracting tissue fluid in a nondestructive painless minimally invasive manner.
Background
Body fluid testing plays an extremely important role in medical work today. Body fluids, which are currently widely used for testing, mainly include blood and urine. For urine, although the urine is simple and easy to obtain, the urine has obvious difference with other body fluid components of the body, so that the detection can have larger deviation; for blood detection, the method is widely applied to clinic, and although the reliability is higher, the method can bring pain and anxiety to patients and risks of vascular damage, wound infection and the like at the same time in view of invasive operation, so that the compliance of the patients is reduced. In addition to the problems associated with blood collection, circulating blood has its limitations in providing healthcare related information. In some cases, the concentration of the drug in the blood has a weak correlation with the concentration at the target site. In this case, as an internal environment where cells directly live, interstitial fluid, advantages directly related to the state of the cells are exhibited. Furthermore, there is only a vascular barrier between the interstitial fluid and the blood, whereas the blood vessels are permeable to most substances other than macromolecular proteins, this anatomical feature makes certain biochemical metabolic index concentrations in the interstitial fluid very close to those in the blood. Tissue fluid occupies about 1/4 of the total body fluid and is mostly distributed on the body surface, so that tissue fluid is extracted to a certain extent more easily than blood extraction in achieving minimally invasive. Based on the analysis, a safer, more effective and more convenient way is needed to be found, tissue fluid is extracted in a minimally invasive and painless way, the accuracy of clinical examination and the compliance of patients are improved, and the occurrence of complications of the traditional method is reduced, so that the patients are better served.
Micropins, which were born in the 70 s of the 20 th century, refer to needle-like structures with diameters less than tens of microns and lengths of 25-2000 μm. By virtue of the extremely fine needle tip, the skin stratum corneum can be easily penetrated under the action of small force, and meanwhile, the micro needle can act on the deep epidermis or the shallow dermis by the short needle body length, so that nerves and blood vessels located in the deep epidermis can not be reached, and the micro needle has the characteristic of painless and minimally invasive (shown in figure 1). As such, microneedles have been widely used in many fields such as disease diagnosis, cosmetics, drug release and treatment of superficial local lesions, and the advent of microneedle technology has made it possible to extract interstitial fluid painless and minimally invasive. The existing microneedles for tissue fluid extraction are in various shapes and have good tissue fluid extraction performance, but the principle of preparing the polymer microneedles is mainly that covalent cross-linking among polymer molecules is firm, so that the subsequent degradation of the microneedles is mainly realized in an enzymology mode. However, the use of biological enzymes such as protease, pancreatin, etc. inevitably has different degrees of negative effects on the structure and properties of the extracts in the microneedles, and even damages, and the state of each substance in the original tissue fluid cannot be perfectly restored without damage, which may reduce the accuracy of subsequent detection, and further affect subsequent clinical diagnosis. At present, a need exists for a microneedle for extracting skin tissue fluid, which has the advantages of simple preparation method, low preparation cost, simple structure and capability of realizing subsequent nondestructive degradation.
Disclosure of Invention
In order to solve the above problems, the present invention provides a microneedle comprising two main body portions, namely a substrate having a patch-like structure and a plurality of solid needle bodies disposed on the substrate; the needle body is in a cone or pyramid structure and is arranged in the patch-shaped substrate, the diameter of the circumcircle of the bottom surface of the needle body is 50-800 mu m, the height is 100-2000 mu m, and the center distance between adjacent needle bodies is 50-1000 mu m.
The invention also provides a preparation method of the microneedle, which comprises the following steps:
(1) Preparing a mixed solution of a microneedle main body raw material and a trace chemical cross-linking agent;
(2) Filling the mixed solution obtained in the step (1) into a microneedle mould;
(3) And (3) standing the system in the step (2), and performing secondary ionic crosslinking, namely, soaking the system in the step (2) in an ionic crosslinking liquid to obtain the target microneedle with obviously enhanced mechanical properties.
Further, in the step (1), the polymer is one or a mixture of several of oxidized sodium alginate, gelatin, oxidized hyaluronic acid, methacrylic anhydride gelatin, cellulose acetate, polyvinyl alcohol, polyether ether ketone, chitosan, hydroxypropyl guanidine gum, polyethylene glycol, polylactic acid, polycaprolactone, dextran, polyether imide, polyamide, polyethylene imine, chitosan, polyacrylamide, polyquaternium-2 diamino urea polymer, polyether amine and dimethylaminopropyl methacrylamide or a copolymer composed of the above polymers.
Further, in the step (1), the chemical crosslinking agent may be one or more of borax, boric acid, sodium metaaluminate, sodium pyrophosphate and potassium pyroantimonate.
Further, in the step (3), the ionic crosslinking solution used for the secondary crosslinking is a higher valence cationic aqueous solution, and the solute thereof may be one or more of calcium chloride, calcium nitrate, barium hydroxide, barium nitrate, barium chloride, magnesium nitrate, magnesium chloride, magnesium sulfate, aluminum nitrate, aluminum chloride, aluminum sulfate, manganese nitrate, manganese chloride, manganese sulfate, zinc nitrate, zinc chloride, zinc sulfate, ferric nitrate, ferric chloride, ferric sulfate, cupric nitrate, cupric chloride, and cupric sulfate.
The invention also provides a method for extracting the tissue fluid in a nondestructive painless minimally invasive way, which comprises the steps of extracting by using the micro-needle, and after the tissue fluid is extracted, placing the micro-needle into the micro-needle degradation liquid integrally to realize the nondestructive degradation of the micro-needle.
Further, the microneedle degradation solution is a low-valence cationic aqueous solution, and the solute of the microneedle degradation solution can be one or more of sodium chloride solution, sodium sulfate, sodium bromide, sodium iodide, sodium nitrate, sodium carbonate, lithium chloride solution, lithium bromide, lithium iodide, lithium sulfate, lithium nitrate, lithium carbonate, potassium chloride solution, potassium bromide, potassium iodide, potassium sulfate, potassium nitrate, potassium carbonate and silver nitrate.
The invention has the following beneficial effects:
(1) The preparation method of the micro-needle can perfectly realize the reversible process of crosslinking-decrosslinking of the micro-needle polymer in an ionic crosslinking mode, thereby ensuring the simple degradation of the micro-needle material and the nondestructive transfer of tissue fluid and protecting the extraction content of the micro-needle. Compared with the traditional covalent crosslinking and enzymatic degradation modes, the invention can reduce the use of biological enzymes, can realize degradation by simple ionic solution, protects the integrity and biological activity of tissue fluid contents, and reduces the cost.
(2) The microneedle provided by the invention has good extraction effect and high detection efficiency. The selected material polymer has good swelling property, and can rapidly and completely extract a sufficient amount of skin tissue fluid. The simple degradation of the extracted material and the nondestructive transfer of tissue fluid also ensure the accuracy of the subsequent biochemical marker detection, and are convenient for assisting the diagnosis of various diseases.
(3) The material used for preparing the micro needle has good biocompatibility, small toxic and side effects and small skin irritation. And the source is wider, and the cost is low.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
The general and microstructure of the microneedle of fig. 1;
FIG. 2 is a swelling image of a microneedle;
FIG. 3 microneedle swelling curve;
FIG. 4 is a graph of compressive stress versus compressive strain for the microneedles of example 1;
FIG. 5 is a graph of rhodamine-agarose penetration effect;
FIG. 6 trypan blue dye microneedle penetration lattice;
FIG. 7 is a photograph of live-dead staining of a microneedle;
FIG. 8 is a graph of microneedle extraction efficiency versus time;
FIG. 9 is a graph of microneedle degradation efficiency over time;
FIG. 10 is a diagram of a microneedle degradation entity;
FIG. 11 is a bar graph of microneedle tissue fluid extraction efficiency;
the left side of fig. 12 is the micro-needle visual view, and the right side is the micro-needle display;
FIG. 13 is a graph of microneedle swelling ratio versus time;
FIG. 14 is a graph of compressive stress versus compressive strain for the microneedles of example 2;
FIG. 15 trypan blue dye microneedle penetration lattice;
FIG. 16 is a photograph of live-dead staining of a microneedle;
FIG. 17 is a graph of microneedle extraction efficiency versus time;
FIG. 18 is a graph of microneedle degradation efficiency over time;
FIG. 19 is a bar graph of microneedle tissue fluid extraction efficiency;
FIG. 20 is a graph of microneedle degradation effects for different crosslinking modes;
FIG. 21 is a graph of microneedle swelling efficiency over time;
FIG. 22 is a graph of microneedle extraction efficiency versus time;
FIG. 23 is a graph of microneedle extraction efficiency versus time;
FIG. 24 is a graph of compressive stress versus compressive strain for the microneedles of comparative example 3.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, with reference to the examples using conventional methods, unless otherwise indicated, and with reference to reagents, either conventional commercial reagents or reagents configured using conventional methods. The detailed description is not to be taken as limiting, but is to be understood as a more detailed description of certain aspects, features, and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Examples 1-9 below are the steps and microneedle parameters for preparing microneedle patches using different materials. Examples 10-20 are characterization of various common performances of the micro-needle, and examples 21-30 are performance verification of nondestructive minimally invasive tissue fluid extraction of the micro-needle.
Example 1
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, 0.045g/ml borax is added after 10% m/v oxidized sodium alginate is stirred uniformly, 10% m/v gelatin is mixed according to a ratio of 5:5 and added into the female die, vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, so that a needle tip is fully poured, the female die is placed into gel at normal temperature, stripping bubbles 5%m/v calcium chloride for 3h (reinforcing the mechanical property of the microneedle through calcium crosslinking) after gradient freezing at-4 ℃, -20 ℃ and-80 ℃, and then microneedle data are obtained through freeze drying: porous microneedles (bottom edge diameter 200 μm, height 800 μm,10 x 10 array) with a pore size of 1000nm and a porosity of 47% (mercury intrusion method) were immersed in 10% m/v sodium chloride solution for 24h after the microneedles were pressed into the agarose hydrogel surface carrying the substance to be detected for 10min (decalcification reaction realizes nondestructive degradation of the extract), and the absorbance of the extracted substance at a specific wavelength could be accurately measured (quantitative detection) by taking the supernatant through a spectrophotometer.
Example 2
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, borax (0.05 g/ml) is added after dextran is oxidized at 12%m/v, the mixture is stirred uniformly, gelatin at 12%m/v is mixed according to a ratio of 5:5 and is added into the female die, after vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, a needle tip is fully poured, the female die is placed into glue at normal temperature, after gradient freezing at-4 ℃, -20 ℃ and-80 ℃, foam 5%m/v magnesium chloride is peeled off for 3h (mechanical property of the microneedle is enhanced through magnesium crosslinking), and then freeze drying is carried out, so that microneedle data are obtained: porous micropins with a pore diameter of 1400nm and a porosity of 50% (mercury porosimetry). The micro-needle is pressed into the agarose water gel surface loaded with the object to be detected for 10min, then the micro-needle is soaked into 10% m/v sodium chloride solution for 24h (magnesium ion removal reaction realizes nondestructive degradation of the extract), and the absorbance (quantitative detection) of the extracted substance under specific wavelength can be accurately measured by a spectrophotometer after supernatant fluid is taken.
Example 3
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, borax (0.045 g/ml) is added after 10% m/v oxidized sodium alginate is stirred uniformly, 10% m/v gelatin is mixed according to a ratio of 5:5 and added into the female die, after vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, a needle tip is fully poured, the female die is placed into glue at normal temperature, after gradient freezing at-4 ℃, -20 ℃, and-80 ℃, peeling bubbles 5%m/v aluminum sulfate are removed for 2h (mechanical property of the microneedle is enhanced through magnesium crosslinking), and freeze drying is carried out, thus obtaining microneedle data: porous micropins with pore size 1200nm and porosity 43% (mercury porosimetry). The micro-needle is pressed into the agarose water gel surface loaded with the object to be detected for 10min, then the micro-needle is soaked into 10% m/v sodium chloride solution for 24h (aluminum ion removal reaction realizes nondestructive degradation of the extract), and the absorbance (quantitative detection) of the extracted substance under specific wavelength can be accurately measured by a spectrophotometer after supernatant fluid is taken.
Example 4
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, borax (0.045 g/ml) is added after 10% m/v oxidized sodium alginate is stirred uniformly, 10% m/v gelatin is mixed according to a ratio of 5:5 and added into the female die, after vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, a needle tip is fully poured, the female die is placed into gel at normal temperature, and after gradient freezing at-4 ℃, -20 ℃ and-80 ℃, stripping bubbles 5%m/v ferric chloride are removed for 3h (mechanical property of the microneedle is enhanced through iron crosslinking), and freeze drying is carried out, so that microneedle data are obtained: porous micropins with pore size 1200nm and porosity 43% (mercury porosimetry). The micro-needle is pressed into the agarose water gel surface loaded with the substance to be detected for 10min, then the micro-needle is soaked into 10% m/v sodium chloride solution for 24h (the iron ion removal reaction realizes the nondestructive degradation of the extract), and the absorbance (quantitative detection) of the extracted substance under a specific wavelength can be accurately measured by taking the supernatant through a spectrophotometer.
Example 5
Firstly, obtaining a Polydimethylsiloxane (PDMS) female die by a microneedle male die through a reverse replication method, adding borax (0.045 g/ml) after oxidizing hyaluronic acid by 10% m/v, uniformly stirring, mixing gelatin by 10% m/v according to a ratio of 5:5, adding the mixture into the female die, vacuumizing for 3-5 times, centrifuging for 10min to fully perfuse a needle tip, placing the mixture at normal temperature to form gel, removing bubbles 5%m/v calcium chloride for 3h after gradient freezing at-4 ℃, -20 ℃ and-80 ℃ (reinforcing the mechanical property of the microneedle through calcium crosslinking), and freeze-drying to obtain microneedle data: the pore diameter is 1600nm, the bottom edge diameter of a porous microneedle with the porosity of 65% (mercury intrusion method) is 280 mu m, the height is 600 mu m, and the array is 15 x 15, after the microneedle is pressed into the surface of agarose gel loaded with a substance to be detected for 10min, the microneedle is soaked in 10% m/v sodium chloride solution for 24h (decalcification reaction realizes nondestructive degradation of an extract), and the absorbance (quantitative detection) of the extracted substance under a specific wavelength can be accurately measured by taking supernatant through a spectrophotometer.
Example 6
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, 2g of chitosan is dissolved in 1% acetic acid solution, dialysis is carried out on the chitosan in a dialysis membrane at room temperature by deionized water, water exchange is carried out for a plurality of times to remove excessive acetic acid (the final pH value is about 6.0), chitosan with the concentration of 10% is prepared, 10% m/v polyvinyl alcohol solution is mixed with the female die, the female die is added, the female die is vacuumized for 3-5 times and centrifuged for 10min to enable a needle tip to be fully poured, the female die is placed into a vacuum drying box for drying at 37 ℃ for 6h, and after gradient freezing at-4 ℃, -20 ℃, -80 ℃, the foam 5%m/v calcium chloride is peeled off for 3h (the mechanical property of the microneedle is enhanced through calcium cross-linking), and then the microneedle data is obtained: the pore diameter is 1400nm, the bottom edge diameter of a porous microneedle with the porosity of 65% (mercury intrusion method) is 200 mu m, the height is about 500 mu m, and the array is 15 x 15- - -after the microneedle is pressed into the surface of agarose water gel loaded with a substance to be detected for 10min, the microneedle is soaked in 10% m/v sodium chloride solution for 24h (decalcification reaction realizes nondestructive degradation of an extract), and the absorbance (quantitative detection) of the extracted substance under a specific wavelength can be accurately measured by taking supernatant through a spectrophotometer.
Example 7
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, 15% m/v polyvinyl alcohol (PVA) is prepared and soaked for 12 hours, then the female die is placed in an oil bath pot at 98 ℃ for stirring for 12 hours, and 30% polyvinylpyrrolidone (PVP) is uniformly mixed according to a ratio of 1:2 and added into the female die, after vacuumizing for 3-5 times, the female die is centrifuged for 10min to fully perfuse a needle tip, the female die is placed in a vacuum drying oven for drying at 37 ℃ for 6 hours, and after gradient freezing at-4 ℃, 20 ℃ and 80 ℃, foam 5%m/v calcium chloride is peeled for 3 hours (the mechanical property of the microneedle is enhanced through calcium crosslinking), and then the microneedle data are obtained through freeze drying: pore size 800nm, pore size 34% (mercury porosimetry) porous microneedle bottom edge diameter 330 μm, high about 770 μm,15 x 15 array- - -after the microneedle is pressed into agarose gel surface of the substance to be detected for 10min, soaking in 10% m/v sodium chloride solution for 24h (decalcification reaction realizes nondestructive degradation of extract), collecting supernatant, and accurately measuring absorbance of the extracted substance under specific wavelength (quantitative detection) by spectrophotometer.
Example 8
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, borax (0.045 g/ml) is added after 10% m/v oxidized sodium alginate is stirred uniformly, 10% m/v gelatin is mixed according to a ratio of 5:5 and added into the female die, after vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, a needle tip is fully poured, the female die is placed into glue at normal temperature, after gradient freezing at-4 ℃, -20 ℃, and-80 ℃, peeling bubbles 5%m/v calcium chloride are removed for 3h (mechanical property of the microneedle is enhanced through calcium crosslinking), and freeze drying is carried out, thus obtaining microneedle data: porous microneedles with a pore diameter of 1000nm and a porosity of 47% (mercury intrusion method), after the microneedles are pressed on the surface of agarose gel for 10min, the microneedles are soaked in a 15% m/v lithium sulfate solution for 24h (decalcification reaction realizes nondestructive degradation of an extract), and the absorbance (quantitative detection) of the extracted substance under a specific wavelength can be accurately measured by taking supernatant through a spectrophotometer.
Example 9
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, borax (0.045 g/ml) is added after 10% m/v oxidized sodium alginate is stirred uniformly, 10% m/v gelatin is mixed according to a ratio of 5:5 and added into the female die, after vacuum pumping is carried out for 3-5 times, centrifugation is carried out for 10min, a needle tip is fully poured, the female die is placed into glue at normal temperature, after gradient freezing at-4 ℃, -20 ℃, and-80 ℃, peeling bubbles 5%m/v calcium chloride are removed for 3h (mechanical property of the microneedle is enhanced through calcium crosslinking), and freeze drying is carried out, thus obtaining microneedle data: porous microneedles with the pore diameter of 1000nm and the porosity of 47% (mercury intrusion method), after the microneedles are pressed on the surface of agarose gel for 10min, the microneedles are soaked in 5%m/v silver nitrate solution for 24h (decalcification reaction realizes nondestructive degradation of an extract), and the absorbance (quantitative detection) of the extracted substance under a specific wavelength can be accurately measured by taking supernatant through a spectrophotometer.
Examples 10-20 the microneedle materials prepared in example 1 will be characterized for their corresponding detection properties, thus fully illustrating the feasibility of the protocol in example 1.
Example 10 characterization of the morphology of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
Characterization of macroscopic morphology the microneedles prepared in example 1 were used to take a macroscopic image of the microneedle patch using an optical camera as shown in fig. 1-a. The prepared microneedle patch is square, and microneedles with the same shape and size are uniformly distributed on one surface of the patch. The microneedles of example 1 were placed on the stage of a confocal microscope, and the microscopic image obtained was as shown in fig. 1-b, with the microneedle array of example 1 being regularly arranged, with the entire conical tip structure being intact and sharper.
EXAMPLE 11 characterization of swelling Property of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
The individual microneedle materials of example 1 were weighed at the zero time point in the dry state (m 0 ) After various periods of time in PBS, the surface liquid was removed with a filter paper sheet, weighed and recorded (m t ). The swelling rate versus time curve is calculated and plotted as shown, and shows that the prepared microneedles are capable of extracting sufficient amounts of liquid in a short time.
EXAMPLE 12 characterization of penetration Properties of microneedle patches for non-destructive, painless minimally invasive extraction of tissue fluids
The microneedles for nondestructive painless minimally invasive tissue fluid extraction prepared in examples 1-4 are placed on a test bench of a universal force measuring machine, the tips of the microneedles face upwards, the stress-dependent relationship is recorded as shown in fig. 4-7, the microneedles are shown to have good mechanical properties, the mechanical strength required for breaking through the stratum corneum can be provided, and then the effective tissue fluid amount can be extracted. The microneedles prepared in examples 2-9 also have the mechanical properties described above.
In vitro penetration experiments A colored agarose gel was prepared by adding 0.05mg/ml rhodamine to the agarose gel (1%m/v). The agarose gel is used for simulating the part below the skin cuticle, a sealing film is covered on the agarose gel to simulate the skin cuticle at the outermost layer, then the micro-needles are attached to the sealing film, the micro-needles are pricked into the agarose with uniform force, the patch is pulled out after 30 seconds, and the micro-needle array distribution on the agarose surface is observed as shown in figure 5.
In a percutaneous experiments, the microneedles prepared in example 1 were inserted into the skin of a flat fresh rat, which was previously prepared for dehairing, and the attachment sites were dip-dyed with 0.4% trypan blue solution after a period of time, and the surface dyeing was washed with 0.9% sodium chloride solution after 10min, and whether or not there were blue spots distributed like a lattice was observed, while the morphological changes before and after the microneedles were observed under a microscope. The surface of the skin of the mice after the action has obvious holes and the rest of the skin is intact, which is observed under an optical microscope, and shows that the porous polymer microneedle can effectively pierce the epidermis of the mice.
Example 13 characterization of biocompatibility of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
The microneedle patch of example 1 was incubated in cell culture medium for 1h, short-term and potential long-term contact of the microneedle patch with skin tissue was simulated, cells were incubated with Human Umbilical Vein Endothelial Cells (HUVEC) for 3 days using the cell culture medium after removal of the microneedle patch, and the proportion of the state of live-dead staining was observed under an inverted fluorescence microscope (compared with the control group), and the histocompatibility of the material was determined as shown in fig. 8, which is an image of live-dead cells of the material of example 1.
Example 14:
adding common biochemical metabolites of human body into agarose solution (1%m/v) to prepare agarose gel, and performing substance extraction in vitro by simulating tissue fluid. The microneedles of example 1 were attached to the gel surface for 5min, 10min, 20min, and 30min, respectively, and then removed, and the mass before and after the attachment of the microneedles was recorded. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is put under shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, so that the content of each substance extracted by the micro needle can be obtained, and the content of each substance is compared with the original concentration of the prepared gel, and the extraction efficiency and the optimal extraction time of the micro needle are verified. The specific data are shown in table 1.
TABLE 1 extraction time and extraction efficiency
Extraction time | 5min | 10min | 20min | 30min | 60min |
Extraction efficiency | 47.4% | 66.9% | 88.4% | 95.3% | 95.5% |
Example 15:
adding common biochemical metabolites of human body into agarose solution (1%m/v) to prepare agarose gel, and performing substance extraction in vitro by simulating tissue fluid. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. Dissolving the attached extracted micro needle in 10% NaCl solution, shaking at 37deg.C for degradation, and respectively taking 200 μl of leaching solution at 1h, 2h, 4h, 6h, 8h, 12h, and 24h, wherein the micro needle is completely dissolved in NaCl solution after 24 h. And measuring the leaching solution under an ultra-micro ultraviolet spectrophotometer to obtain the content of each substance extracted by the micro needle, comparing the content with the original concentration of the prepared gel, and verifying the release efficiency of the micro needle. The specific data are shown in table 2.
TABLE 2 degradation time and degradation efficiency
Degradation time | 1h | 2h | 4h | 6h | 8h | 12h | 18h | 24h |
Degradation efficiency | 8.2% | 15.4% | 25.3% | 40.6% | 62.4% | 76.5% | 88.6% | 99.2% |
Example 16:
glucose (10 mg/mL) was added to the agarose solution (1%m/v) to prepare a glucose-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, so that the content of glucose extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on glucose reaches 99.71%, as shown in figure 11.
Example 17:
albumin (5 mg/mL) was added to an agarose solution (1%m/v) to prepare a protein-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the content of albumin extracted by a microneedle can be obtained, and is compared with the original concentration of prepared gel, and the detection efficiency of the microneedle on protein reaches 99.67%, as shown in figure 11.
Example 18:
uric acid (0.5 mg/mL) was added to an agarose solution (1%m/v) to prepare uric acid-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the wavelength is 293nm, the content of uric acid extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on uric acid reaches 99.82%, and the detection efficiency is shown in figure 11.
Example 19:
ascorbic acid (0.2 mg/mL) was added to the agarose solution (1%m/v) to prepare an ascorbic acid-agarose gel, and the simulated substance extraction was performed in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the wavelength is 260nm, the content of uric acid extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on ascorbic acid reaches 99.56%, and the detection efficiency is shown in figure 11.
Example 20
Glucose (10 mg/mL), albumin (5 mg/mL), uric acid (0.5 mg/mL) and ascorbic acid (0.2 mg/mL) were added to an agarose solution (1%m/v), and the resulting mixture was mixed to prepare agarose gel, and the simulated substance was extracted in vitro. And (5) attaching the gel on the surface of the gel for 30min by using a microneedle, and taking off. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under a trace ultraviolet spectrophotometer at specific wavelength of each substance to obtain the content of each substance extracted by a microneedle, and the content is compared with the original concentration of prepared gel, wherein the detection efficiency of the microneedle on glucose, albumin, uric acid and ascorbic acid respectively reaches 99.56%, 99.45%, 99.78% and 99.44%, and the detail is shown in figure 11.
Examples 21-31 the oxidized dextran and polyacrylamide cross-linked microneedle materials prepared in example 2 were correspondingly characterized for detection performance, thus fully demonstrating the feasibility of the protocol in example 2.
Example 21 characterization of the morphology of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
Characterization of macroscopic morphology the microneedles prepared in example 2 were placed in a petri dish and a macroscopic image of the microneedle patch was taken by an optical camera, as shown in fig. 12, the microneedle patch was square, and microneedles of the same shape and size were uniformly distributed on one side of the patch. The microneedle of example 2 was placed on the stage of a stereoscopic microscope, and a microscopic image was obtained as shown in fig. 12, and the whole of the microneedle of example 2 was a cone-like needle tip with a complete and sharp structure.
EXAMPLE 22 characterization of swelling Property of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
The individual microneedle materials of example 2 were weighed at the zero time point in the dry state
(m 0 ) After various periods of time in PBS, the surface liquid was removed with a filter paper sheet, weighed and recorded (m t ). The swelling rate versus time curve is calculated and plotted as shown, and shows that the prepared microneedles are capable of extracting sufficient amounts of liquid in a short time. The microneedles of examples 2 to 9 also possess the above properties.
EXAMPLE 23 characterization of penetration Properties of microneedle patches for non-destructive painless minimally invasive extraction of tissue fluids
1. The microneedle for nondestructive painless minimally invasive tissue fluid extraction prepared in the example 2 is placed on a test bench of a universal force measuring machine, the tip of the microneedle faces upwards, the relationship recorded to stress following the strain is shown in fig. 14, the mechanical property of the microneedle is shown to be good, the mechanical strength required for breaking through the stratum corneum can be provided, and the effective tissue fluid amount can be extracted. The microneedles prepared in examples 2 to 9 also have the above mechanical properties.
2. In a percutaneous experiments, the microneedles prepared in example 2 were inserted into the skin of a flat fresh rat, which was previously prepared for dehairing, and the attachment sites were dip-dyed with 0.4% trypan blue solution after a period of time, and the surface dyeing was washed with 0.9% sodium chloride solution after 10min, and whether or not there were blue spots distributed like a lattice was observed, while the morphological changes before and after the microneedles were observed under a microscope. The surface of the skin of the mice after the action has obvious holes and the rest of the skin is intact, which is observed under an optical microscope, and shows that the porous polymer microneedle can effectively pierce the epidermis of the mice.
EXAMPLE 24 characterization of the biocompatibility of microneedle patches for non-destructive, painless minimally invasive extraction of tissue fluids
The microneedle patch of example 2 was incubated in cell culture medium for 1h, short-term and potential long-term contact of the microneedle patch with skin tissue was simulated, and after removal of the microneedle patch, cells were incubated with Human Umbilical Vein Endothelial Cells (HUVEC) for 3 days using the cell culture medium, and the proportion of the state of live-dead staining was observed under an inverted fluorescence microscope (compared with the control group), and the histocompatibility of the material was determined as shown in fig. 16, which is an image of live-dead cells of the material of example 2.
Example 25:
adding a colored substance rhodamine into agarose solution (1%m/v) to prepare agarose gel, and performing substance extraction in vitro by simulating tissue fluid. The microneedles of example 1 were attached to the gel surface for 5min, 10min, 15min, 20min, 30min, and 1h, respectively, and then removed, and the mass before and after the attachment of the microneedles was recorded. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, so that the content of each substance extracted by the micro needle can be obtained, and the content of each substance is compared with the original rhodamine concentration in the prepared gel, and the extraction efficiency and the optimal extraction time of the micro needle are verified. The specific data are shown in Table 3.
TABLE 3 extraction time and extraction efficiency
Extraction time | 5min | 10min | 15min | 20min | 30min | 60min |
Extraction efficiency | 52% | 69.2% | 75.6% | 89.4% | 96.3% | 98.8% |
Example 26:
adding a colored substance rhodamine into agarose solution (1%m/v) to prepare agarose gel, and performing substance extraction in vitro by simulating tissue fluid. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. Dissolving the attached extracted micro needle in 10% NaCl solution, shaking at 37deg.C for degradation, and respectively taking 200 μl of leaching solution at 1h, 2h, 4h, 6h, 8h, 12h, and 24h, wherein the micro needle is completely dissolved in NaCl solution after 24 h. And measuring the leaching solution under an ultra-micro ultraviolet spectrophotometer to obtain the content of each substance extracted by the micro needle, comparing the content with the original concentration of the prepared gel rhodamine, and verifying the release efficiency of the micro needle. The specific data are shown in table 4.
TABLE 4 degradation time and degradation efficiency
Degradation time | 1h | 2h | 4h | 8h | 12h | 18h | 24h | 36h |
Degradation efficiency | 12.4% | 25.8% | 39.3% | 61.6% | 78.5% | 89.6% | 95.4% | 98.9% |
Example 27:
rabbit actin (5 mg/mL) was added to agarose solution (1%m/v) to prepare a rabbit actin-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, so that the content of glucose extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on glucose reaches 99.21%, and the content is shown in figure 19.
Example 28:
bovine albumin (5 mg/mL) was added to the agarose solution (1%m/v) to prepare bovine serum albumin-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the content of albumin extracted by a microneedle can be obtained, and is compared with the original concentration of prepared gel, and the detection efficiency of the microneedle for protein reaches 99.43 percent, which is shown in figure 19.
Example 29:
uric acid (0.5 mg/mL) was added to an agarose solution (1%m/v) to prepare uric acid-agarose gel, and the simulated substance was extracted in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the wavelength is 293nm, the content of uric acid extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on uric acid reaches 99.87%, and the detection efficiency is shown in figure 19.
Example 30:
ascorbic acid (0.2 mg/mL) was added to the agarose solution (1%m/v) to prepare an ascorbic acid-agarose gel, and the simulated substance extraction was performed in vitro. Taking down the gel after the gel is attached to the surface of the gel for 30min by using a microneedle, and recording the mass before and after the microneedle is attached. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under an ultra-micro ultraviolet spectrophotometer, the wavelength is 260nm, the content of uric acid extracted by a microneedle can be obtained, and compared with the original concentration of prepared gel, the detection efficiency of the microneedle on ascorbic acid reaches 99.76%, and the detection efficiency is shown in figure 19.
Example 31
Rabbit actin (5 mg/mL), bovine serum albumin (5 mg/mL), uric acid (0.5 mg/mL) and ascorbic acid (0.2 mg/mL) were added to agarose solution (1%m/v), and the mixture was mixed to prepare agarose gel, and the simulated substance was extracted in vitro. And (5) attaching the gel on the surface of the gel for 30min by using a microneedle, and taking off. The attached and extracted micro-needle is dissolved in 10% NaCl solution, and is subjected to shaking at 37 ℃ for degradation, and after 24 hours, the micro-needle can be observed to be completely dissolved in the NaCl solution. 200 mu L of the leaching solution is measured under a trace ultraviolet spectrophotometer at specific wavelength of each substance to obtain the content of each substance extracted by a microneedle, and the content is compared with the original concentration of prepared gel, wherein the detection efficiency of the microneedle against rabbit actin, bovine serum albumin, uric acid and ascorbic acid respectively reaches 98.60%, 99.11%, 99.45% and 99.54%, and the detail is shown in figure 19.
Comparative example 1 comparison of degradation Rate
The effect of the amounts of 0.1% borax and 5% borax on the cross-linking-degradation of the materials was compared and the results are shown in table 5. Dissolving 10% m/v oxidized sodium alginate, adding 0.1% m/v borax and 5%m/v borax respectively, stirring, dissolving uniformly, and mixing with 15% m/v gelatin at a ratio of 5:5. After standing for gel formation, the gel was placed in a 10% NaCl solution at 37 ℃ for degradation by shaking, and the mass change data of the microneedle material are shown below. The material of covalent cross-linking (5%m/v borax) is difficult to degrade, while trace borax added into the material of ionic cross-linking (0.1% m/v borax) does not change the cross-linking property of the material and does not influence the degradation of the material.
TABLE 5 influence of borax amounts of 0.1% and 5% on crosslinking-degradation of materials
Comparative example 2
10g of gelatin was dissolved in 100ml of PBS solution and stirred with a magnetic stirrer in a water bath at 60 ℃. After complete dissolution, a further 8ml of MA solution (mass ratio 8%) were added. Reacting at 50deg.C for three hours, dialyzing in deionized water at 50deg.C for one week, and drying to obtain GelMA precursor (lyophilized at-20deg.C and stored in a 4 deg.C refrigerator). Weighing GelMA at 15% m/v, dissolving in deionized water at 50deg.C, adding 10mg photoinitiator (2 ml system) at 60deg.C, casting the system solution onto PDMS mold, centrifuging for 5min at 3500rpm to fill gel on needle tip, and adding a solvent at 500mW/cm 2 The ultraviolet crosslinking is carried out for 40-60 min under the irradiation of ultraviolet light, then the ultraviolet crosslinking is carried out in 10% m/v sodium chloride solution for 24h, the swelling capacity is 200% lower than that of the embodiment 1, and the preparation of the research template is only suitable for laboratory research, and has high cost and is not simple.
Comparative example 3
Firstly, a microneedle male die is used for obtaining a Polydimethylsiloxane (PDMS) female die through a reverse replication method, urea, N-dimethylformamide, glycine and 2-ethoxyethanol are used for modifying silk fibroin, after vacuuming for 3-5 times, centrifuging is carried out for 10min to enable a needle tip to be fully poured, polyacrylic acid is used as a backing, the backing is placed and formed at normal temperature, and after gradient freezing at-4 ℃ and-20 ℃, stripping bubbles 5%m/v calcium chloride are removed for 3h (mechanical properties of the microneedle are enhanced through iron crosslinking), and freeze drying is carried out, so that microneedle data are obtained: porous microneedles (bottom diameter 250 μm, height 550 μm,10 x 10 array) with pore size 500-700nm and porosity 15% (mercury intrusion). After the microneedles are pressed into the agarose water gel surface loaded with substances to be detected (rabbit actin and bovine albumin) for 10min, the corresponding buffer solution is added to be mixed, the microneedles are soaked by using a solution containing 0.25% elastase (dissolved in PBS), the solution is hydrolyzed for 30-60 min at the temperature of pH=8.5 and the temperature of T=45 ℃, the concentration of the substances to be detected is detected by using an ninhydrin colorimetric method, and the extraction efficiency is calculated, so that the single detection rate and the mixed detection rate of various proteins of the microneedle material in the comparative material and the microneedle material in the embodiment are shown in figure 17, and the extraction rate of the microneedles of the invention is obviously higher than that of the comparative material, which accords with the theory that the proteases which are supposed in advance have certain damage effect on the proteins in the microneedles when degrading the microneedles, the nondestructive extraction of the targets of the present patent can be visually highlighted through the comparative example.
Comparative example 4
Obtaining a Polydimethylsiloxane (PDMS) female die by a microneedle male die through a reverse replication method, adding borax (0.045 g/ml) after 10% m/v oxidized sodium alginate, uniformly stirring, mixing with 10% m/v gelatin according to a ratio of 5:5, adding the mixture into the female die, vacuumizing for 3-5 times, centrifuging for 10min to fully perfuse a needle tip, placing the mixture into gel at normal temperature, and stripping the gel after gradient freezing at-4 ℃, -20 ℃, -80 ℃ to obtain microneedle data: porous microneedles with a pore size of 2000nm and a porosity of 73% (mercury porosimetry). The compression modulus is significantly lower than in example 1 and does not provide the mechanical strength required to break through the stratum corneum, see in detail fig. 24.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (7)
1. A microneedle comprising two main body parts, namely a substrate with a patch-like structure and a plurality of solid needle bodies positioned on the substrate; the needle body is in a cone or pyramid structure and is arranged in the sheet-shaped substrate, the diameter of the circumcircle of the bottom surface of the needle body is 50-800 mu m, the height is 100-2000 mu m, and the center distance between adjacent needle bodies is 50-1000 mu m.
2. A method of preparing the microneedle according to claim 1, comprising the steps of:
(1) Preparing a mixed solution of a microneedle main body raw material and a trace chemical cross-linking agent;
(2) Filling the mixed solution obtained in the step (1) into a microneedle mould;
(3) And (3) standing the system in the step (2), and performing secondary ionic crosslinking, namely, soaking the system in the step (2) in an ionic crosslinking liquid to obtain the target microneedle with enhanced mechanical properties.
3. The method according to claim 2, wherein in the step (1), the main raw material comprises a polymer: oxidized sodium alginate, gelatin, oxidized hyaluronic acid, methacrylic anhydride gelatin, cellulose acetate, polyvinyl alcohol, polyether ether ketone, chitosan, hydroxypropyl guanidine gum, polyethylene glycol, polylactic acid, polycaprolactone, dextran, polyetherimide, polyamide, polyethyleneimine, chitosan, polyacrylamide, polyquaternium-2 diamino urea polymer, polyether amine, dimethylaminopropyl methacrylamide or a copolymer composed of the above polymers.
4. The method according to claim 2, wherein in the step (1), the chemical crosslinking agent is one or more of borax, boric acid, sodium metaaluminate, sodium pyrophosphate, and potassium pyroantimonate.
5. The method according to claim 2, wherein in the step (3), the ionic crosslinking solution used for the secondary crosslinking is an aqueous solution containing a cation of a higher valence state, and the solute thereof is one or more of calcium chloride, calcium nitrate, barium hydroxide, barium nitrate, barium chloride, magnesium nitrate, magnesium chloride, magnesium sulfate, aluminum nitrate, aluminum chloride, aluminum sulfate, manganese nitrate, manganese chloride, manganese sulfate, zinc nitrate, zinc chloride, zinc sulfate, ferric nitrate, ferric chloride, ferric sulfate, cupric nitrate, cupric chloride, and cupric sulfate.
6. A method for minimally invasive extraction of tissue fluid without damage and pain, which is characterized in that the microneedle according to claim 1 is used for extraction, and after the tissue fluid extraction is finished, the microneedle is wholly placed in a microneedle degradation solution to degrade materials.
7. The method according to claim 6, wherein the microneedle degradation solution is a low-valence cationic aqueous solution, and the solute is one or more of sodium chloride, sodium sulfate, sodium bromide, sodium iodide, sodium nitrate, sodium carbonate, lithium chloride, lithium bromide, lithium iodide, lithium sulfate, lithium nitrate, lithium carbonate, potassium chloride, potassium bromide, potassium iodide, potassium sulfate, potassium nitrate, potassium carbonate, and silver nitrate.
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