The present invention relates to a microneedle and a sensor for detecting nitrogen monoxide, including the same.
In general, a microneedle is used for delivery of active materials such as a drug and a vaccine in vivo, detection of an analyte in vivo, and biopsy. As a method of using a microneedle, a form in which a certain number of holes are formed on the skin by using a microneedle device such as a roller to which the microneedle is attached, and then a drug is applied thereon, a form in which the surface of a microneedle is coated with an active ingredient (effective ingredient) to allow the active ingredient to be administered simultaneously with perforation of the skin, a form in which when injected with a microneedle using a polymer (a biodegradable polymer or a water-soluble polymer), an active material included in the microneedle is decomposed or dissolved in the skin and diffused, and the like are generally used.
Meanwhile, it is most important to early diagnose cancer which is characterized by metastasis, and an endoscope is used as the most common and easiest method in order to prevent cancer. Various kinds of endoscopes for diagnosing cancer more accurately have been abundantly developed, and representative examples thereof include an electronic endoscope, a capsule endoscope, a 3D endoscope, and the like. After cancer is primarily classified by using images obtained from an endoscope using a dye staining method, the tissue of interest is collected, and then it is determined whether cancer is malignant or not. However, for the current method, it is essential to perform a tissue examination in order to accurately determine whether the tumor is malignant or not, and there are problems in that it takes a long time to determine whether the tumor is malignant through the tissue examination, and images obtained through the staining method of the endoscope may be sometimes incorrectly analyzed, thereby leading to the occurrence of misdiagnosis.
The present invention has been made in an effort to solve the aforementioned problems and provide a sensor which is capable of diagnosing cancer by a non-invasive method within a short period of time by using a microneedle.
The present invention relates to a microneedle, a sensor for detecting nitrogen monoxide, including the microneedle, a medical apparatus including the microneedle, and a manufacturing method thereof.
The present invention provides a microneedle in which a microneedle base; an adhesive polymer layer; a conductive polymer layer; and a nitrogen monoxide bonding molecule layer including iron ions are sequentially stacked.
The present invention also provides a sensor for detecting nitrogen monoxide, the sensor including: the microneedle; and an electrode.
The present invention also provides a sensor for diagnosing cancer, the sensor including: the microneedle; and an electrode.
The present invention also provides an endoscope including the microneedle, the sensor for detecting nitrogen monoxide, or the sensor for diagnosing cancer.
The present invention also provides a method for manufacturing a microneedle, the method including: forming an adhesive polymer layer on a microneedle base by mixing the microneedle base with an adhesive polymer; forming a conductive polymer layer on the adhesive polymer layer through a solution process by bringing the adhesive polymer layer into contact with a conductive polymer solution; and forming a nitrogen monoxide bonding layer on the conductive polymer layer by bringing the conductive polymer layer into contact with a nitrogen monoxide bonding molecule layer including iron ions.
The microneedle of the present invention may detect whether nitrogen monoxide is present or not by using electrochemical principles. Further, a change in concentration of nitrogen monoxide may be sensed in real time. The effects of detecting nitrogen monoxide may be used to diagnose cancer and forecast the size and growth degree of tumor.
The present invention provides a microneedle in which a microneedle base; an adhesive polymer layer; a conductive polymer layer; and a nitrogen monoxide bonding molecule layer including iron ions are sequentially stacked.
The present invention also provides a method for manufacturing a microneedle, the method including: forming an adhesive polymer layer on a microneedle base by mixing the microneedle base with an adhesive polymer; forming a conductive polymer layer on the adhesive polymer layer through a solution process by bringing the adhesive polymer layer into contact with a conductive polymer solution; and forming a nitrogen monoxide bonding layer on the conductive polymer layer by bringing the conductive polymer layer into contact with a nitrogen monoxide bonding molecule layer including iron ions.
The microneedle of the present invention is comprised of a microneedle base, an adhesive polymer layer, a conductive polymer layer, and a nitrogen monoxide bonding molecule layer. The conductive polymer layer and the nitrogen monoxide bonding molecule layer are for detecting nitrogen monoxide, and the adhesive polymer layer is necessary for stably constituting the conductive polymer layer and the nitrogen monoxide bonding molecule layer.
In the present specification, the microneedle base forms a basic framework of a microneedle, exhibiting the shape of the microneedle. The microneedle of the present invention is completed by forming additional coating layers on the microneedle base. The microneedle base may be manufactured by a common publicly known method according to the constituent material. For example, a microneedle base composed of a biodegradable polymer may be formed by putting the biodegradable polymer into a mold for a microneedle, adding heat to the mold, and cooling the mold. In the case of a microneedle base composed of aluminum oxide, aluminum nickel, nickel oxide or stainless steel and the like, a microneedle may be formed by using an etching method, and in the case of a microneedle base composed of stainless steel, a microneedle may be formed by an etching method or a metal mold casting method.
In one specific exemplary embodiment, the microneedle base may be one or more selected from the group consisting of polyvinyl alcohol, polyethylene glycol, polylactide, polyglycolide, polyethylene oxide, polydioxanone, polyphosphazene, polyanhydride, polyamino acid, polyacrylate, polyacrylamide, polyurethane, polysiloxane, polyvinylpyrrolidone, polycaprolactone, polymethylmethacrylate, polyethylene, polyamide, polydimethylsiloxanes, polyester, polyorthoester, polycyanoacrylates, polyphosphazenes, polyvinylchrolide, polymethylpentene, polynitrobenzyl, polyaminoester, cellulose acetate butyrate, cellulose triacetate, polyethylene terephthalate, Teflon (polytetrafluoroethylene), stainless steel, silicon, silicon oxide, aluminum, aluminum oxide, nickel oxide, and SU-8 (an epoxy-based negative type photoresist), but is not limited thereto. In one specific exemplary embodiment, the microneedle base may be composed of a biodegradable polymer, and the biodegradable polymer may be a hydrophobic polymer. In one specific exemplary embodiment, polycaprolactone (PCL) may be used as a base of the microneedle, and polycaprolactone is a biodegradable material and has very high biostability. Further, strength of the microneedle may be appropriately adjusted by adjusting the molecular weight of polycaprolactone. In one specific exemplary embodiment, the molecular weight of polycaprolactone may be 4,000 to 10,000 kDa, 5,000 to 9,000 kDa, 6,000 to 85,000 kDa or 7,000 to 8,300 kDa, but is not limited thereto.
An adhesive polymer layer may be formed on the microneedle base by mixing the microneedle base with an adhesive polymer. In one specific exemplary embodiment, the adhesive polymer may be chitosan, silk, collagen, fibronectin, vitronectin, rubber, or polydopamine, but is not limited thereto. For example, the adhesive polymer may include a catechol group, and may be polydopamine. Dopamine is a polymer having adhesive properties and high biostability and causes self-polymerization on the hydrophobic surface of a polycaprolactone microneedle according to the oxidation reaction, so that a dopamine polymer is grown on the surface of the microneedle base composed of polycaprolactone, thereby allowing the surface of the microneedle base to be coated. Accordingly, the surface of the microneedle exhibits hydrophilicity, and is simultaneously modified into a surface having very high adhesion. Such a surface modification facilitates coating of PEDOT to be subsequently used, and adhesion is excellent, and thus stability of the microneedle is improved.
In one specific exemplary embodiment, subjecting the microneedle base to UV treatment or ozone plasma treatment may be additionally included before forming the adhesive polymer layer on the microneedle base. The UV treatment or the ozone plasma treatment is a surface modification method publicly known in the art, and the surface coating capability of a conductive polymer layer to be added may be improved through the treatment. For example, the UV treatment may be performed by using a UVO cleaner apparatus to perform the UV for 25 to 35 minutes or 30 minutes, the ozone plasma treatment may be performed by using a reactive ion etching (RIE) apparatus (SNTEK BSC5004) to treat the ozone plasma under a vacuum of 5×10−6 torr or less under the oxygen atmosphere at a power of 100 for 50 to 70 seconds or 60 seconds.
In one specific exemplary embodiment, the conductive polymer may be polyacetylene, polyaniline, polypyrrole, polythiophene, poly(1,4-phenylenevinylene), poly(1,4-phenylene sulfide), poly(fluorenylene ethynylene), polyisothianaphthene, polythienylene vinylene, polyphenylene vinylene, polyphenylene sulfide, polyhexylthiophene, PEDOT, or derivatives thereof, but is not limited thereto. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a bluish polymer, and a conductive polymer which is innocuous in vivo, has good biostability, and is easy to be handled in the process. Due to these characteristics, the PEDOT is used for a channel or an electrode of a sensor in various fields such as a biosensor, a semiconductor, and a solar cell. The chains of a PEDOT polymer have a number of pi electrons, form a pi-pi bond with pi electrons which other molecules have, and thus affect the conductivity of the PEDOT polymer when doped with an n-type or p-type dopant. The PEDOT is present in a solution state, mixed with a surfactant PSS in an aqueous solution, and in order to use a polymer having a molecular weight within a suitable range, the PEDOT is filtered by a filter, and then coated on the microneedle coated with dopamine, which becomes hydrophilic, through a solution process.
In one specific exemplary embodiment, the solution process may be performed by immersing a microneedle base, on which an adhesive polymer layer is formed, in a conductive polymer solution, and drying the microneedle base.
In one specific exemplary embodiment, a nitrogen monoxide bonding molecule including iron ions may be a porphyrin ring or a hemin molecule, which has pi electrons in the core thereof. The hemin molecule having a structure similar to hemoglobin is an unpaired orbital material having trivalent iron ions in the core, and rapidly captures nitrogen monoxide in vivo to cause a nitrosylation. Extra pi electrons present in the center of the porphyrin ring of the hemin molecule are bonded to other pi electrons to form a pi-pi bond, and are bonded to a number of extra pi electrons present in the PEDOT polymer chains, thereby forming a channel of the sensor. When the PEDOT polymer is used as a channel of the sensor, a small amount of the PEDOT polymer is decomposed in the air due to the hydrophilic tendency which is an inherent property of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate 1000 (PEDOT: PSS 1000) which is used for coating, but when one layer of a hydrophobic molecule hemin is coated on the PEDOT layer, the channel is prevented from being damaged and stability of the microneedle is increased by entirely covering a PEDOT channel.
The microneedle may be formed on a microneedle pad. The microneedle pad means a plate on which the microneedle is formed, and the function of the sensor may be imparted to the microneedle by depositing an electrode on the microneedle pad. For example, for the microneedle of the present invention, 10 needles in both width and length directions are present in a square form on the microneedle pad, and thus, a total of 100 needles may be formed.
Accordingly, the present invention also provides a sensor for detecting nitrogen monoxide, including the microneedle and an electrode. The present invention also provides a method for manufacturing a sensor for detecting nitrogen monoxide, the method including depositing an electrode on the microneedle pad in which the microneedle is formed.
In one specific exemplary embodiment, the electrode may be composed of one or more selected from the group consisting of nickel, chromium, titanium, gold, silver, and platinum. Nickel, chromium, titanium and the like may be used as an electrode for being adhered to a gold or silver electrode.
In one specific exemplary embodiment, the electrode may include a reference electrode, a working electrode, and a counter electrode.
The sensor for detecting nitrogen monoxide according to the present invention may diagnose cancer by detecting the amount of nitrogen monoxide around the cancer cell. Accordingly, the present invention provides a sensor for diagnosing cancer, including the microneedle and the electrode.
In most cancers such as esophageal, gastric, colorectal, and skin cancers, an inducible nitric oxide synthase gene is excessively expressed for the process of metastasis and apoptosis of cancer cells, and a large amount of nitrogen monoxide is secreted from the cancer cells by the inducible nitric oxide synthase gene. Nitrogen monoxide is always secreted from the tissues around the cancer cell by 1,000 times or more than from the normal cells, and thus, the concentration of nitrogen monoxide is maintained at a high concentration at a micromole level for several days. Nitrogen monoxide is a radical molecule in a very unstable state, and is rapidly diffused along the blood vessels, the lymph vessels, and tissues when generated in vivo. When nitrogen monoxide combines with hemoglobin in the blood in the in vivo environment, nitrogen monoxide is captured by the iron ions of the hemoglobin, and annihilated.
That is, the amount of nitrogen monoxide present around the cancer cells is an important biomarker which may diagnose cancer. The sensor including the microneedle according to the present invention may measure the amount of nitrogen monoxide, which is a biomarker produced in a large amount from malignant tumor cells, in real time, so that it may be determined whether a subject is afflicted with malignant tumor in a biological fluid by a non-invasive method.
In one specific exemplary embodiment, the cancer may be skin cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, uterine cancer, or breast cancer, but is not limited thereto.
When nitrogen monoxide is captured by iron of hemin molecules in a channel in which the PEDOT and the hemin molecule form a pi-pi bond, the resistance of the PEDOT varies as the density of pi electrons of the hemin molecule forming a pi bond with PEDOT is changed.
More specifically, the trivalent iron ions of the hemin molecule tend to receive one electron and become stabilized because the 4s orbital of the iron ions is in an empty state. Therefore, the trivalent iron ions become more stable while being bonded to a number of pi electrons present in the polymer chains of PEDOT to form a pi-pi bond. The PEDOT polymer is present in a p-type doping state by a pi-pi bond with the hemi molecules, and an electron carrier, which provides conductivity to the PEDOT polymer, becomes a positive hole flowing along the chains. In this case, when nitrogen monoxide composed of radicals approaches the hemin molecule, the trivalent iron ions of the hemin molecule accept electrons of nitrogen monoxide which forms a stronger bond, the electron density of iron ions is partially packed from nitrogen monoxide and dispersed, and the electron density is also transferred to the polymer chains of PEDOT, thereby increasing the electron density of PEDOT. Accordingly, a positive hole, which is an electron carrier of the p-type doped PEDOT polymer chains, is bonded to an electron by an additional inflow of electrons, and the density of the electron carrier is reduced while the function as the electron carrier is offset, thereby increasing the resistance of the sensor channel. That is, the presence and absence and amount of nitrogen monoxide may be detected depending on the degree of reduction in resistance according to the change in current of the microneedle measured in real time.
In one specific exemplary embodiment, the height of the microneedle of the present invention may be 300 to 1,000 micrometers, 400 to 900 micrometers, 500 to 800 micrometers, and 550 to 750 micrometers. When the height of the microneedle is within the range, the microneedle does not touch the nerves and causes no pain to a subject while being applied to the subject, and may sufficiently pass through the skin in the subcutaneous layer and access the portion around cancer, which is exposed to the dermal layer.
The manufacturing of the sensor for detecting nitrogen monoxide according to the present invention may additionally include performing a waterproof treatment, except for the microneedle part, after depositing the electrode on the microneedle pad. In one specific exemplary embodiment, the waterproof treatment may be performed by coating the sensor with a polymer for waterproof treatment, such as a silicon-based polymer, a parylene-based polymer, a non-conductive plastic, or a hydrophobic polymer. The sensor may be subjected to waterproof treatment by being coated with the polymer for waterproof treatment except for the PEDOT coating region for detecting nitrogen monoxide. When the portion other than the channel of the microneedle sensor is covered with the polymer for waterproof treatment, a large noise may be reduced because the electrode portion other than the channel does not directly touch an aqueous solution containing nitrogen monoxide or the skin, and the detection efficiency may be improved because the sensor has a form of sensing nitrogen monoxide only sensed in the channel.
The silicon polymer includes sinylon, polyurethane, epoxy, polydimethylsiloxane, decamethyl cyclopentasiloxane, and the like, but is not limited thereto. The parylene-based polymer includes Parylene-A, Parylene-B, Parylene-C polymers, and the like, but is not limited thereto.
The microneedle or the sensor for detecting nitrogen monoxide according to the present invention may be safely applied to a living organism by using materials which are innocuous to the living organism. For example, the sensor of the present invention may also be applied as it is to the skin, and may also be used while being attached to an endoscope for diagnosing cancer.
Accordingly, the present invention also provides an endoscope including the microneedle or the sensor for detecting nitrogen monoxide.
The microneedle of the present invention has a small size of 0.5 cm2 or less, and may be manufactured along with the endoscope. The sensor for detecting nitrogen monoxide may be electrically connected and attached to an endoscope which is typically used for diagnosing cancer. As the method for attaching the sensor for detecting nitrogen monoxide to the endoscope, a typical method publicly known may be used without limitation.
For example, the sensor for detecting nitrogen monoxide may be applied in vivo as a hose-type or a capsule-type depending on the kind of endoscope, and when the sensor is introduced in vivo, the channel part of the microneedle is introduced in vivo while being surrounded by a protective film, and the amount of nitrogen monoxide may be detected by dissolving the protective film immediately before sensing a specific tissue, and then pricking the tissue with the needle.
The microneedle or the sensor for detecting nitrogen monoxide according to the present invention may replace an existing tissue examination, which is cumbersome and takes a long period of time when the in vivo tumor is diagnosed by an endoscope. The endoscope including the microneedle or the sensor for detecting nitrogen monoxide according to the present invention may determine a malignant tumor conveniently and at low costs by confirming a position presumed to be an in vivo tumor cell through the endoscope, and measuring the concentration of nitrogen monoxide around the confirmed tissue as a change in current flow in an aqueous solution state in real time. That is, it is possible to directly distinguish in vivo whether the tumor is malignant or benign by confirming the position suspected to be a tumor by a dye method, and then inserting the microneedle in vivo instead of collecting the tissue. Further, the prognosis of a tumor may be rapidly and easily forecast by forecasting the size and growth degree of a tumor according to the degree of reduction in resistance based on a change in current of the microneedle, which is measured in real time. Since the microneedle may be applied to various tumors, the microneedle of the present invention may be mounted to various kinds of endoscopes, and variously diagnose characteristics of the tumors.
In one specific exemplary embodiment, the endoscope includes a gastroscope, a bronchial endoscope, a colonofiberscope, an esophageal endoscope, a duodenum endoscope, a bladder endoscope, a celioscope, a thoracic cavity endoscope, or a cardiac endoscope, and the like, but is not limited thereto.
Hereinafter, the present invention will be described in detail with reference to the following Examples. However, the following Examples are only for exemplifying the present invention, and the content of the present invention is not limited by the following Examples.
A PDMS mold having 10 needle patterns in both width and length directions was filled with 0.5 g of beads of polycaprolactone having a molecular weight of 8,000 kDa, and then, the beads were dissolved in an oven at 180° C. under vacuum conditions for approximately 3 hours. After the mold was removed from the oven, the microneedle base cooled at normal temperature was separated from the mold. Dopamine hydrochloride at a concentration of 2 mg·ml−1 was dissolved in a tris buffer at a pH of 8.5 and a concentration of 1.0 mM. And then, the polycaprolactone microneedle base was put into the solution, the resulting solution was stirred at 37° C. for 24 hours, and the surface of the microneedle base was coated with dopamine. In order to prepare a PEDOT channel, a solution mixed with PEDOT:PSS 1000 was filtered by a 0.45 mm filter, and then a solution process was performed by covering the microneedle base doped with dopamine with the solution, and drying the microneedle base at 37° C. in an oven. The surface of the microneedle was entirely coated with poly(methyl methacrylate) (PMMA), and then, only the channel portion of the microneedle was exposed by using e-beam lithography. Next, hemin molecules dissolved in an organic solvent of dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml were placed on the PEDOT channel, and settled for 24 hours. The PEDOT channel was washed three times each with DMSO and an isopropyl alcohol solution to remove hemin molecules, which were not bonded, from the PEDOT channel, thereby manufacturing a dopamine-PEDOT-hemin molecule channel.
In order to manufacture three electrodes, titanium, palladium, and gold were sequentially deposited on the microneedle. AZ4620 photoresist was spin-coated on the surface of the microneedle, the microneedle was baked at 65° C. in an oven for 20 minutes, and then only a square-shaped site on which an electrode was to be deposited was selectively etched. Next, titanium, palladium, and gold were deposited in 10 nm, 10 nm, and 50 nm, respectively, by using an e-beam evaporator. And then, an unnecessary photoresist was all etched by washing the microneedle with isopropyl alcohol and distilled water. Finally, in order to subject a portion except for the PEDOT channel for detection to water proof treatment, the microneedle except for a channel site (5×5 mm) coated with PEDOT was coated with PDMS, and then cured at 60° C. in an oven, thereby manufacturing a microneedle sensor (1×1 cm). The configuration of the manufactured microneedle sensor is illustrated in
In order to detect nitrogen monoxide in vitro, diethylamine NONOate sodium salt was dissolved in 10 mM of a PBS buffer having a pH of 7.4, in which 10 mM of NaOH had been dissolved, and the resulting solution was used as a supply source of nitrogen monoxide. Before the NONOate sodium salt was dissolved, oxygen was completely removed by bubbling 10 mM of the PBS buffer solution having a pH of 7.4, in which 10 mM of NaOH had been dissolved, with nitrogen for 2 hours, and then the NONOate sodium salt was dissolved immediately before a nitrogen monoxide detection test was performed by the sensor, thereby providing nitrogen monoxide.
After the RAW 264.7 macrophage cells were grown in a DMEM cell culture solution for about one day, the first group was a control and was not treated with any reagent, the second group was a group rich in nitrogen monoxide and was subjected to treatment of 0.5 ug/ml of lipopolysaccharide (LPS) with a cell culture solution in order to cause nitrogen monoxide to be produced in a large amount, and the third group was a group in which the amount of nitrogen monoxide had been reduced and was subjected to treatment with aminoguanidine at a concentration of 100 mM along with 0.5 ug/ml of lipopolysaccharide.
In order to construct a skin cancer mouse model, B 16F10 cells with a density of 1×107 were put into both sides on the back of an SKH-1 mouse. After 2 weeks, when the skin cancer cells with a size of 0.5 cm3 were sufficiently grown, the current was measured by thoroughly cleaning the skin on the cancer cells with ethanol, and inserting the microneedle into the skin.
1-1) Surface Test of Microneedle Sensor
In order to confirm whether up to the needle end portion of the microneedle manufactured in Preparation Example 1 had been coated well with the conductive PEDOT channel, the surface of the microneedle sensor was observed by an optical microscope and a scanning electron microscope. Furthermore, in order to observe the hemin molecules coated on the PEDOT channel, various signals emitted by interaction of the sample surface with electron beam were analyzed by using an EDX detector which may analyze the constituent elements and relative amount of a material, and it was confirmed whether hemin molecules were present or not by detecting characteristic X-ray to qualitatively analyze chemical components having a micro structure. Further, in order to confirm the iron composition and chemical bonding state of the hemin molecule on the surface of the sample by measuring the photoelectron energy emitted by allowing characteristic X-ray to be incident to the surface of the microneedle, XPS was performed.
1-2) Analysis Result
The result is illustrated in
As a result of observation by a scanning electron microscope, it was confirmed that the surface of the initial microneedle (microneedle base) composed of polycaprolactone was clearly present (
Accordingly, it could be seen from the images obtained by the optical microscope and the scanning electron microscope that the PEDOT channel was not separated from the microneedle and up to the end portion of the needle was coated well only when the microneedle coated with dopamine was subjected to a solution process of the PEDOT channel.
As illustrated in
Further, as illustrated in
1-1) Measurement of Strength of Microneedle
In order to confirm mechanical properties of the microneedle, pressure (stress) was measured according to the measurement of strength, failure stress, and displacement length (strain). A graph in change of forces was obtained at a speed of 0.5 mm/min according to the compression length by using an Instron eXpert 760 mechanical tester (ADMET), and a compression constant was obtained by converting the graph into a graph related to stress and strain. In addition, holes produced on the skin by substantially inserting the microneedle into the skin of the mouse were observed by an optical microscope.
1-2) Analysis Result
As illustrated in
Furthermore, as illustrated in
As illustrated in
1-1) Measurement of Current According to Voltage Using Circulating Current Method
For the surface analysis of the sensor, the circulating current was measured at each coating step of dopamine, PEDOT, and hemin molecules by using a CHI 832 workstation (Shanghai Chenhua, China). As an electrolyte, a PBS buffer solution with a pH of 7.4 was selected, and the current measurement was performed at a scan rate of 50 mV/s. A graph of measuring the circulating current of the microneedle sensor was obtained according to the scan rate.
Further, an oxidation and reduction peak of nitrogen monoxide was analyzed by treating the PBS buffer solution with nitrogen monoxide at each concentration, and measuring the circulating current.
In order to measure the resistance of the surface of the microneedle sensor, electrochemical impedance spectra (EIS) were measured by using a CHI 660 electrochemical workstation. The impedance spectra were measured in the PBS buffer solution with a pH of 7.4 under an ac of 5 mV according to the frequency in the range from 0.1 Hz to 100 KHz.
1-2) Analysis Result
As illustrated in
As illustrated in
As illustrated in
As illustrated in
1-1) Observation of Surface of PEDOT Channel Before and after Inserting Microneedle into Skin
The channel surface of the microneedle was observed through a scanning electron microscope before and after the microneedle was inserted into the skin of the mouse. Further, it was confirmed through the confirmation of the oxidation-reduction peak of hemin molecules by measuring the circulating current under the same conditions as in Experimental Example 3 after washing the microneedle sensor with a DMSO organic solvent for 5 days that hemin molecules could be bonded well to the PEDOT channel, and a change in graph was observed by measuring the circulating current 50 consecutive times under the same conditions as described above.
1-2) Analysis Result
As a result of observing the surface state of the microneedle before and after the microneedle was inserted into the skin by a scanning electron microscope, it could be confirmed as illustrated in
As illustrated in
1-3) Quantification of Hemin Molecules Bonded to PEDOT Channel
The number of moles of hemin molecules attached to the microneedle was calculated. First, a standard of the concentrations of hemin molecules was obtained according to the intensity of absorbance at 405 nm by obtaining the UV spectra at each concentration of hemin molecules dissolved in DMSO. Next, the microneedle coated with the PEDOT channel was immersed in 0.1 ml of a solution of hemin molecules at an initial concentration of 1 mg/ml for one day, and then the concentration of hemin molecules left in the solution was calculated by using the UV spectrum to measure the intensity of absorbance.
1-4) Analysis Result
The concentration of hemin molecules was calculated according to the following equations by using the UV spectrum.
S
hemin
−N
hemin
/S
pEDOT
N
hemin
=N
hemin0
−N
hemin1=(Chemin0−Chemin1)×V
In this case, Shemin means a concentration of hemin molecules bonded on the PEDOT channel per surface area, Nhemin means the total number of hemin molecules bonded to the PEDOT channel, Nhemin0 means the number of initial hemin molecules in the solution before the microneedle is loaded, and Nhemin1 means the number of hemin molecules left in the solution after the microneedle is loaded. The difference between Nhemin0 and Nhemin1 indicates the number of hemin molecules bonded to the microneedle. SPEDOT is an area of a PEDOT channel and 1 cm2, Chemin0 is 1 mg/ml which is an initial concentration of a hemin group, and Chemin1 is a concentration of hemin molecules obtained from the UV spectrum. Since Shemin=0.91 nm−2 through the equations, it could be seen that the concentration of hemin molecules was a concentration at which the hemin molecules are bonded on the PEDOT channel as a monolayer, considering that the diameter of hemin molecules is 0.5 nm.
In order to measure the degree of cytotoxicity of the microneedle coating layers, the MTT reagent, which measures the activity of cells, was used. The Raw 146.7 macrophage cells were grown in the same number each on the polycaprolactone microneedle, the microneedle coated with dopamine, the microneedle coated with both dopamine and the PEDOT channel, and the microneedle coated with even dopamine, the PEDOT channel, and a hemin group, the cells were completely attached thereto after 1 to 2 days, and then 150 ml of the MTT solution filtered by a 0.2 mm filter at a concentration of 0.5 mg ml−1 was administered to the cells. The needles were warmed at 37° C. in an oven for about 2 hours, and the solution on the needle was completely removed. And then, 200 μl of DMSO was put into the microneedles to completely dissolve cells which turned purple, and then the absorbance was measured in a wavelength range of 540 nm by using the ELISA microplate reader (molecular devices, Versamax).
As illustrated in
1-1) Real-Time In Vitro Nitrogen Monoxide Detection Test at Each Concentration
As illustrated in
1-2) Analysis Result
As illustrated in
1-1) Measurement of Reaction to In Vivo Various Polysaccharides and Proteins by Microneedle
Before the microneedle was substantially applied in vivo, it was examined whether the conductivity of the microneedle channel was changed by various kinds of polysaccharides and proteins with a high electron density present in vivo. Solutions in which galactose, glucose, a trivalent iron ion, peroxide, an ovalbumin protein, lysozyme, and a bovine serum albumin protein are dissolved at a concentration of about 1 uM were sequentially flowed into the microneedle sensor, and finally, nitrogen monoxide at 1 and 2 uM was sequentially flowed into the microneedle sensor.
1-2) Analysis Result
As illustrated in
1-3) Real-Time Detection Test of Nitrogen Monoxide in Cell Culture Solution (Dulbecco's Modified Eagle Medium (DMEM))
A real-time detection test of nitrogen monoxide was performed in a cell culture solution including various proteins, amino acids, vitamins, and inorganic salts by replacing the PBS solution with DMEM in the same manner as in the real-time detection test of nitrogen monoxide performed in Experimental Example 6.
1-4) Analysis Result
As illustrated in
1-1) Conversion of Concentration of Nitrogen Monoxide
In order to examine the substantial amount of nitrogen monoxide emitted from the three groups of the macrophage cells of Preparation Example 4, the absorbance of the solution in a wavelength range of 540 nm was obtained depending on the concentration of nitrogen monoxide emitted from the quantified diethylamine NONOate sodium salt by using a grease reagent test. The absorbance was measured at a total of 11 concentrations by continuously diluting the amount of nitrogen monoxide emitted by using diethylamine NONOate sodium salt by ½ from 250 uM. 100 ul of the solution of diethylamine NONOate sodium salt at various concentrations was mixed with 100 ul of a grease reagent, and 10 minutes later, a standard was obtained based on the intensity value of the absorbance in a wavelength range of 540 nm.
1-2) Analysis Result
As illustrated in
1-1) Real-Time Detection of Nitrogen Monoxide Generated from Macrophage Cells
During the metabolism process of the living RAW 264.7 macrophage cells, a maximum nano molarity of nitrogen monoxide was generated. When a cell medium which grows the macrophage was treated with a lipopolysaccharide, nitrogen monoxide at a concentration which is 1,000 times or higher than a usual concentration by increasing the differentiation of an inducible nitric oxide synthase (iNOS) which is one of the enzymes which produce nitrogen monoxide from cells. Further, when the cell medium was treated with aminoguanidine along with the lipopolysaccharide, the lipopolysaccharide was suppressed to again reduce the amount of nitrogen monoxide emitted from the cells.
As illustrated in
1-2) Quantification of Amounts of Nitrogen Monoxide Generated from Three Groups of Cells
In order to analyze the production degree of nitrogen monoxide from the macrophage according to the concentrations of lipopolysaccharide (LPS) and aminoguanidine, a grease reagent test was performed. 10,000 macrophages were cultured in a 6-well plate for cell culture for one day, and then treated with lipopolysaccharide at 0, 0.25, 0.5, 1, 2, and 4 μg/ml, a grease test was performed one day later, and the absorbance produced from the UV spectrum was converted by using the nitrogen monoxide concentration relationship according to the absorbance obtained in
1-3) Analysis Result
As a result, as illustrated in
As illustrated in
1-1) Real-Time Measurement of Nitrogen Monoxide by Using Mouse with Induced Skin Cancer
The microneedle which was bonded to hemin molecules, and the microneedle which was not bonded to hemin molecules were pricked on the site on which the skin cancer cells were grown, thereby measuring the real-time current change. As a control, a general mouse without induced skin cancer was used.
1-2) Analysis Result
As illustrated in
Number | Date | Country | Kind |
---|---|---|---|
10-2014-0122889 | Sep 2014 | KR | national |