WO2017007271A1 - Patch cutané de diagnostic enduit d'aptamères et à base de micro-aiguilles - Google Patents

Patch cutané de diagnostic enduit d'aptamères et à base de micro-aiguilles Download PDF

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Publication number
WO2017007271A1
WO2017007271A1 PCT/KR2016/007410 KR2016007410W WO2017007271A1 WO 2017007271 A1 WO2017007271 A1 WO 2017007271A1 KR 2016007410 W KR2016007410 W KR 2016007410W WO 2017007271 A1 WO2017007271 A1 WO 2017007271A1
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WIPO (PCT)
Prior art keywords
aptamer
microneedle
patch
skin
coated
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PCT/KR2016/007410
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English (en)
Korean (ko)
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손인식
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주식회사 넥스모스
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Publication of WO2017007271A1 publication Critical patent/WO2017007271A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to aptamer coated microneedle based diagnostic skin patches.
  • Skin diseases represent a major healthcare challenge in the world today. With over one million skin cancers diagnosed each year in the United States (National Cancer Research Institute, www.cancer.gov), predicting and diagnosing skin diseases is important in terms of their management. Current diagnostic methods rely primarily on visual observation and biopsy. However, detection methods that rely on visual observation are not necessarily effective in diagnosing a skin condition or disease and do not detect risk or disease until clinical signs appear. In addition, invasive methods such as biopsies increase the likelihood of infection as well as trauma to the test subject. In addition, the method must be performed by a physician in order to be safe and usually does not usually provide abundant cell samples on the skin surface, the cells involved in the reaction.
  • noninvasive methods of diagnosing and monitoring skin conditions and diseases represent an important means for the care of patients and for evaluating the efficacy of existing and new therapeutics, skin care products and skin care regimens.
  • the method can provide important information regarding the genetic predisposition of the test subject to the occurrence of skin disease, as well as specific genetic changes based on the skin condition of the test subject. Identifying these genetic changes can be important in identifying potential drug targets and preventive measures and in determining whether a person actually responds to a particular therapeutic agent, skin care product or regimen.
  • detection and diagnostic methods are important for evaluating the safety of such treatments, products, and measures.
  • composition of the substance of the skin is changed in various disease states as well as local skin diseases. It is known that various substances such as lipids, structural proteins, inflammatory substances, nucleic acids, and metabolites can be detected in the skin according to the disease state. In addition to atopic dermatitis, melanoma, and bacterial inflammation of the skin, biomarkers are being analyzed for various diseases such as Alzheimer's disease, Parkinson's disease, breast cancer, cardiovascular disease, diabetes, and drug addiction. However, in most cases very invasive skin biopsy is used.
  • the present invention has been made in view of the above problems and needs, and an object of the present invention is to provide a patch for use in the diagnosis of various diseases.
  • the present invention comprises the steps of: a) binding an amine group to an aptamer; b) attaching a silanol to the surface of the microneedle tip by plasma oxidation, and then adding a hydroxyl; Apylmer-coated microneedle comprising the step of silanizing with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) to a group, and then bonding an amine group bonded to the aptamer to an epoxy group included in the 3-GPTMS. It provides a method for manufacturing a base skin diagnostic patch.
  • the present invention is treated with HNO 3 on the surface of the polycarbonate microneedle, and then reduced to NaBH 4 to finally combine the amine group, polyethylene glycol (PEG) is bonded to the carboxyl group at one end and the aptamer at the other end
  • PEG polyethylene glycol
  • a method for preparing aptamer-coated microneedle-based skin diagnostic patch comprising the step of combining using NHS (N-Hydroxysuccinimide), and EDC [1-ethyl-3- (3'-dimethylaminopropyl) carbodiimide] as a catalyst .
  • the present invention also provides an aptamer-coated microneedle-based skin diagnostic patch prepared by the method of the present invention.
  • the skin diagnostic patch is characterized in that the aptamer is attached to the microneedle tip surface and the diagnostic component can be attached to the end of the aptamer, but is not limited thereto.
  • the diagnostic component is preferably a protein, peptide, DNA or RNA, but is not limited thereto.
  • the present invention provides a diagnostic skin that effectively detects various biomarkers present in intracellular interstitial cells by coating a microneedle surface of about 200 um in length that can penetrate the stratum corneum of the skin and lead to epidermis. It's a patch.
  • biomarkers that can be collected in the intracellular interstitial tissue are secreted by the infecting bacteria or infectious organisms in various infectious diseases.
  • malaria bacteria or proteins released by the bacteria are representative proteins.
  • PfHRP2 PfHRP2
  • the virus itself can be detected, and in the case of various degenerative brain diseases, for example, the Tau protein is increased in Parkinson's or Alzheimer's disease and can be used as a marker.
  • Synuclein can also be used as an important diagnostic marker for Parkinson's disease
  • Alymoid beta can be used as a diagnostic marker for Alzheimer's disease
  • various cancer markers can be used as a biomarker that can be collected.
  • microneedles In connection with the production of aptamer-coated microneedle of the present invention, various types of microneedles have been developed for effective delivery of drugs through brain-machine interfaces (BMIs) or skin and have been tested for measurement of EEG through actual scalp.
  • BMIs brain-machine interfaces
  • microneedles or microprojection arrays of various materials and lengths are being manufactured.
  • the types of chemicals used in the microneedle are as follows. Polyurethane (PU), Polypropylene (PP), Polyethylene (PE), Polystyrene (PS), Poly (methyl methacrylate) (PMMA), Polydimethylsiloxane (PDMS), Polycarbonate (PS), Liquid crystal polymer (LCP).
  • An aptamer is a method that detects a specific substance by using a three-dimensional structure of single strand DNA or RNA, which is similar to an antigen-antibody reaction, but because the substance is much smaller, aptamers can be attached to the ends or holes of microneedles. There is an advantage that it can.
  • aptamers for various biomarkers can be attached at once (similar to gene chips) to detect multiple kinds of materials at the same time (Multiplexing).
  • a gene chip a genome capable of detecting more than 10,000 DNA or RNA can be combined on a nail size chip, so the skin patch can also be used as a biomarker chip by activating a microneedle as an aptamer for various substances.
  • a method for detecting a specific substance by using a three-dimensional structure of single strand DNA or RNA called aptamer is similar to an antigen-antibody reaction, but the size of the substance is much smaller and relatively large.
  • the advantage is that a large number of aptamers can be bonded to the microneedle tip surface.
  • aptamers for various biomarkers can be attached at one time, multiple substances can be detected at the same time (Multiplexing), and microneedle tip-based skin patches can be used as protein chips using aptamers.
  • 1 is a structure according to the fabrication of the microneedle and the skin layer using PDMS
  • 3 is a method of attaching silanol to the surface of the microneedle tip by plasma oxidation, silanizing the hydroxyl group with 3-GPTMS, and then combining the amine group of PEG coupled with the aptamer to the epoxy group included in 3-GPTMS.
  • Figure 5 is a method of coating a polycarbonate microneedle surface with an aptamer bonded to one end of the PEG.
  • A After treating HNO3 on the surface of PC microneedle, it is reduced to NaBH4 to finally bind the amine group. Thereafter, PEG, which combines a carboxyl group at one end with an aptamer at the other end, is treated with PC-NH2 along with EDC / NHS to complete the PEG-aptamer functionalized PC microneedle.
  • B Identification of NH2-coupled PC via ATR-FTIR.
  • C Analysis of the amount of aptamer attached to the surface of the PC using fluorescence tagged anti-sense oligonucleotide.
  • the microneedle surface is treated with polyethylene glycol (PEG), and then aptamer is attached to the PEG end to construct the device.
  • PEG polyethylene glycol
  • Silanol (SiOH) is formed through plasma oxidation of the surface of PDMS constituting the microneedle.
  • silanizing this Hydroxyl group with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group aptamer-PEG can be formed on the surface of the microneedle by combining the amine group (Fig. 4) of PEG with an aptamer combined with this expoxy group. (FIG. 3).
  • the surface of the PC microneedle was treated with nitric acid, followed by electrophilic substitution, and reduced nitro groups to prepare PC-NH 2 with primary amine groups.
  • the aptamer bound to PEG having a carboxyl group is bound thereto using N-Hydroxysuccinimide (NHS) and 1-ethyl-3- (3′-dimethylaminopropyl) carbodiimide (EDC) as a catalyst (FIG. 5A). That is, HNO3 is treated on the surface of the PC microneedle and then reduced to NaBH4 to finally bind the amine group. Thereafter, PEG, which combines a carboxyl group at one end with an aptamer at the other end, is treated with PC-NH2 along with EDC / NHS to complete the PEG-aptamer functionalized PC microneedle.
  • NHS N-Hydroxysuccinimide
  • EDC 1-ethyl-3- (3′-dimethylaminopropyl

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Microbiology (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medical Informatics (AREA)
  • Surgery (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un patch cutané de diagnostic enduit d'aptamère et à base de micro-aiguilles. Le patch de la présente invention présente des avantages : la taille d'un matériau est considérablement plus petite qu'un matériau anticorps, et un grand nombre d'aptamères peut être lié aux surfaces d'extrémité d'un nombre relativement important de micro-aiguilles. De plus, le patch de la présente invention permet la fixation d'aptamères pour plusieurs types de biomarqueurs en même temps, et peut ainsi détecter plusieurs types de matériaux en même temps (multiplexage), de sorte qu'une pointe de patch cutané à base de micro-aiguilles peut également être utilisé en tant que puce à protéines à l'aide d'un aptamère.
PCT/KR2016/007410 2015-07-09 2016-07-08 Patch cutané de diagnostic enduit d'aptamères et à base de micro-aiguilles WO2017007271A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2015-0097854 2015-07-09
KR20150097854 2015-07-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018124327A1 (fr) * 2016-12-28 2018-07-05 주식회사 넥스모스 Procédé de fabrication de timbre cutané de diagnostic fondé sur des micro-aiguilles revêtu d'aptamères et timbre

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020081597A1 (en) * 2000-03-31 2002-06-27 Genentech, Inc. Compositions and methods for detecting and quantifying gene expression
US20110160069A1 (en) * 2008-05-23 2011-06-30 The University Of Queensland Analyte detection using a needle projection patch
KR20130060009A (ko) * 2011-11-29 2013-06-07 삼성전자주식회사 표적 물질 검출 및 분리 장치, 및 이를 이용한 표적 물질 검출 및 분리 방법
US20140287942A1 (en) * 2012-12-14 2014-09-25 The Scripps Research Institute Methods and Devices for Detection and Acquisition of Biomarkers
KR101502204B1 (ko) * 2012-01-27 2015-03-13 경희대학교 산학협력단 앱타머 칩을 이용한 표적 단백질의 검출 및 정량 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020081597A1 (en) * 2000-03-31 2002-06-27 Genentech, Inc. Compositions and methods for detecting and quantifying gene expression
US20110160069A1 (en) * 2008-05-23 2011-06-30 The University Of Queensland Analyte detection using a needle projection patch
KR20130060009A (ko) * 2011-11-29 2013-06-07 삼성전자주식회사 표적 물질 검출 및 분리 장치, 및 이를 이용한 표적 물질 검출 및 분리 방법
KR101502204B1 (ko) * 2012-01-27 2015-03-13 경희대학교 산학협력단 앱타머 칩을 이용한 표적 단백질의 검출 및 정량 방법
US20140287942A1 (en) * 2012-12-14 2014-09-25 The Scripps Research Institute Methods and Devices for Detection and Acquisition of Biomarkers

Non-Patent Citations (1)

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Title
KAMRA, T. ET AL.: "Covalent Immobilization of Molecularly Imprinted Polymer Nanoparticles Using an Epoxy Silane", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 445, 7 January 2015 (2015-01-07), pages 277 - 284, XP029165030 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018124327A1 (fr) * 2016-12-28 2018-07-05 주식회사 넥스모스 Procédé de fabrication de timbre cutané de diagnostic fondé sur des micro-aiguilles revêtu d'aptamères et timbre

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