WO2023224312A1 - Multifunctional biomedical fluorescent nanoparticles using dopamine or dopamine analogs, preparation method therefor and use thereof - Google Patents

Multifunctional biomedical fluorescent nanoparticles using dopamine or dopamine analogs, preparation method therefor and use thereof Download PDF

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WO2023224312A1
WO2023224312A1 PCT/KR2023/006388 KR2023006388W WO2023224312A1 WO 2023224312 A1 WO2023224312 A1 WO 2023224312A1 KR 2023006388 W KR2023006388 W KR 2023006388W WO 2023224312 A1 WO2023224312 A1 WO 2023224312A1
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dopamine
fluorescent nanoparticles
pfnps
present
hydrogel
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PCT/KR2023/006388
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French (fr)
Korean (ko)
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이창수
이경관
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한국생명공학연구원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels

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  • the present invention relates to multifunctional biomedical fluorescent nanoparticles using dopamine or dopamine analogues, a method of manufacturing the same, and uses thereof. More specifically, fluorescent nanoparticles made of a polymer of dopamine and its analogues, a method of manufacturing the same, and imaging the same. It relates to various uses for application to photothermal therapy, etc.
  • polymers in the medical field include support materials such as implants or artificial organs, artificial blood vessels, intraocular lenses, artificial joints, prosthetics, suture materials, extracorporeal therapy materials, etc., or blood perfusion, blood oxygenators, catheters, blood tubing. Other support materials such as those used in tubing, wound and burn covering materials, splints, contact lenses, etc.
  • support materials such as implants or artificial organs, artificial blood vessels, intraocular lenses, artificial joints, prosthetics, suture materials, extracorporeal therapy materials, etc., or blood perfusion, blood oxygenators, catheters, blood tubing.
  • Other support materials such as those used in tubing, wound and burn covering materials, splints, contact lenses, etc.
  • polymers have been used in the development of nanoparticle delivery systems and controlled-release delivery systems, and research has been conducted to target drug delivery to a desired location. Additionally, polymers have been found to be used in applied biological therapies such as transdermal drug delivery patches, microspheres, enzymes, and cell immobilization.
  • Dopamine is a type of neurotransmitter that acts as a messenger between nerve cells and is a type of amine containing the chemical components of catechol or catechin.
  • Polydopamine (PDA) is produced when dopamine (DA) is polymerized, and the polydopamine-coated surface introduces various functional groups by covalently adsorbing molecules with amine (-NH 2 ) or thiol groups.
  • DA dopamine
  • -NH 2 amine
  • thiol groups thiol groups
  • it can be coated on various supporting surfaces such as various metals, silicon oxide, iron oxide, stainless steel, Teflon, and polystyrene, and has excellent adhesion.
  • Korean Patent No. 10-1539389 B1 discloses a cofactor regeneration method using polydopamine-derived plasmonic nanohybrid, and polydopamine produced by adding polydopamine, metal nanoparticles, and dye to the substrate
  • An artificial photosynthesis method is disclosed in which cofactors are regenerated using a dopamine-induced plasmonic nanohybrid and useful substances are produced through an oxidoreductase reaction using the regenerated cofactors.
  • one aspect of the present invention is to provide a method for producing fluorescent nanoparticles.
  • Another aspect of the present invention is to provide fluorescent nanoparticles with excellent bioapplicability.
  • Another aspect of the present invention is to provide an imaging composition comprising fluorescent nanoparticles.
  • Another aspect of the present invention is to provide a hydrogel containing fluorescent nanoparticles.
  • a method for producing fluorescent nanoparticles comprising mixing dopamine or a dopamine analog with C 1 -C 5 alkylenediamine in a basic solvent.
  • fluorescent nanoparticles that are polymers of dopamine or a dopamine analog are provided.
  • an imaging composition capable of imaging multiple areas including the fluorescent nanoparticles of the present invention is provided.
  • a hydrogel comprising the fluorescent nanoparticles of the present invention, PVA, and water.
  • the fluorescent nanoparticles of the present invention have a simple manufacturing process, and when implemented as bioadhesive films and hydrogels using them, they not only have excellent adhesion to biological tissues, but are highly useful for bioimmobilization and monitoring through fluorescence.
  • the photothermal conversion efficiency due to irradiation of near-infrared wavelengths is high, so it can be applied to photothermal therapy, for example, cancer cell-specific photothermal treatment.
  • FIG 1 schematically shows the manufacturing process of fluorescent nanoparticles (PFNPs) using dopamine and dopamine analogues.
  • Figure 2 shows (a) TEM, (b) c) FT-IR spectrum, (d) Zeta potential analysis results.
  • Figure 3 shows the results of X-ray photoelectron spectroscopy (XPS narrow) spectrum analysis of PFNPs synthesized from dopamine and dopamine analogues.
  • FIG. 4 shows the emission fluorescence spectrum of the fluorescent nanoparticles (PFNPs) of the present invention according to structural differences.
  • Figure 5 shows (a) the fluorescence lifetime spectrum of the fluorescent nanoparticles (PFNPs) of the present invention and (b) the change in fluorescence spectrum according to the polymerization reaction time.
  • Figure 6 shows fluorescence images of the polydopamine-based fluorescent nanoparticles (LVD, NPP, HDA, DA, EPP fNPs) of the present invention in MRC-5 cells.
  • Figure 7 shows an in vivo fluorescence image of the polydopamine-based fluorescent nanoparticles of the present invention.
  • Levodopa (LVD) fluorescent nanoparticles are blue
  • nornephpyrine (NPP) fluorescent nanoparticles are green
  • 6-hydroxydopamine is yellow to light green
  • dopamine is orange
  • epinephrine Each can display a red fluorescence image.
  • Figure 8 shows the cytotoxicity results of (a) LVD, (b) NPP, (c) HDA, (d) DA, and (e) EPP fNPs based on polydopamine fluorescent nanoparticles of the present invention on MRC-5 cells. .
  • Figure 9 (a) schematically shows the manufacturing process of the PFNPs-PDMS film
  • Figure 9 (b) shows an image of the prepared PFNPs-PDMS film.
  • Figure 10 schematically shows the mold manufacturing process for testing the bioadhesion strength of the PFNPs-PDMS film.
  • Figure 11 schematically shows the evaluation method of the tack separation experiment of the PFNPs-PDMS film.
  • Figure 12 (a) shows a method for calculating adhesion energy using UTM
  • Figures 12 (b) and (c) show the results of comparing the adhesion strength of the PFNP-PDMS film measured from UTM.
  • Figure 13 shows a fluorescence image of a mouse implanted with PFNPs-PDMS.
  • Figure 14 shows the results of histological toxicity analysis of the PFNPs-PDMS film through H&E staining.
  • Figure 15 schematically shows the hydrogel production and photothermal effect mechanism using the fluorescent nanoparticles of the present invention.
  • Figure 16 shows photothermal properties of PFNPs-PVA hydrogel prepared from (a) PVA-hydrogel and dopamine analog (b) LVD, (c) NPP, (d) HDA, (e) DA, and (f) EPP. This shows the conversion effect.
  • Figure 17 shows the in vivo photothermal conversion effect of a hydrogel using the fluorescent nanoparticles of the present invention.
  • Figure 18 shows (a) temperature change of mice injected with PFNPs-PVA hydrogel over time according to near-infrared laser irradiation, (b) change in body weight of mice following photothermal treatment for 14 days, and (c) change in cancer cell volume.
  • Figure 19 shows the H&E staining results of cancer cells after photothermal treatment using PFNPs-PVA hydrogel.
  • Figure 20 shows the H&E staining results of heart, lung, liver, spleen, and kidney cells after photothermal treatment using PFNPs-PVA hydrogel.
  • Figure 21 shows the results of fluorescence imaging observed for 15 days by transplanting the DA-PDMS film and EPP-PDMS film into the epidermis of a balb/c nude mouse and living epidermal tissue, (a) balb/c nude mouse Photographs before and after fNPs-PDMS implantation, (b) biofluorescence imaging (Cy 3 wavelength region) of mice injected with DA fNPs PDMS and EPP fNPs PDMS films, and (c) mice injected with DA fNPs PDMS and EPP fNPs PDMS films. This shows a graph of quantitative fluorescence intensity change in mice (15 days, 3-day intervals).
  • a method for producing fluorescent nanoparticles based on dopamine and/or its analogs is provided.
  • the fluorescent nanoparticles provided by the present invention may be referred to interchangeably as PFNPs (Polydopamine-based Fluorescent Nanoparticles) or fNPs.
  • the method for producing fluorescent nanoparticles of the present invention includes the step of mixing dopamine or a dopamine analog with C 1 -C 5 alkylenediamine in a basic solvent.
  • the dopamine analog may be at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine.
  • the C 1 -C 5 alkylenediamine includes methylenedianim, ethylenediamine, propylenediamine, etc., and is preferably ethylenediamine.
  • the method for producing fluorescent nanoparticles of the present invention is to mix at least one component of dopamine and its analogs in an aqueous solution containing C 1 -C 5 alkylenediamine and ammonia for 1 to 24 hours at room temperature, e.g. For example, it can be prepared by reacting for 1 to 12 hours.
  • the aqueous solution may additionally contain alcohol such as ethanol.
  • the basic solvent may be an ammonia aqueous solution, and ammonia is preferably used in an amount of 2 to 3% in the aqueous solution. If the ammonia content is less than the above range, polymerization through self-assembly may be difficult. there is.
  • 50 to 200 mg, for example 100 to 150 mg, of the dopamine or its analogue may be mixed with 1 to 10 mL, for example 3 to 7 mL of C 1 -C 5 alkylenediamine, 10 mL to 50 mL, for example 20 to 7 mL.
  • an alkaline aqueous solution mixed with 40 mL of distilled water, 5 to 15 mL, for example, 7 to 11 mL of ethanol, and 0.5 to 5 mL of ammonia water, for example, ammonia water (NH 4 OH) with a concentration of 2 to 3%.
  • the reaction can be carried out at room temperature for 1 to 8 hours, for example, 1 to 5 hours.
  • the alkaline aqueous solution of the present invention preferably has a pH of 9 to 11, for example, 9.5 to 10.5.
  • the C 1 -C 5 alkylenediamine is preferably used in a volume ratio of 0.5 mL to 5 mL with respect to 30 mL of (aqueous) solution in which dopamine and/or its analogs are dissolved, and C 1 -C 5 If the alkylenediamine is less than the above range, there is a problem of low yield, and if it exceeds the above range, the weight average molecular weight of the polymer may be low.
  • fluorescent nanoparticles which are polymers of dopamine and/or dopamine analogs
  • the fluorescent nanoparticles of the present invention are multifunctional composite nanoparticles that can be applied to the biomedical field as a fluorescent material, and enable multiple bio-imaging of animal cells using low toxicity and multicolor (red, green, blue) fluorescence.
  • the fluorescent nanoparticles of the present invention may be nanoparticles with a particle diameter of 50 to 300 nm.
  • the particle size of each nanoparticle is 50 to 200 nm and can be adjusted depending on the polymerization time and the amount of monomer added.
  • the dopamine analog may be at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine, for example, the fluorescence
  • the nanoparticles may be a combination of fluorescent nanoparticles containing at least one of levodopamine polymer, 6-hydroxydopamine polymer, and epinephrine polymer particles.
  • an imaging composition containing fluorescent nanoparticles is provided.
  • an imaging composition capable of imaging multiple areas including multiple types of fluorescent nanoparticles may be provided.
  • These multiple types of fluorescent nanoparticles exhibit the characteristic of emitting fluorescence of different wavelengths such as red, green, and blue depending on their different molecular structures, and based on this, multiple fluorescence imaging of animal cells is possible.
  • the plurality of types of fluorescent nanoparticles may be a combination of fluorescent nanoparticles containing at least one of levodopamine polymer, 6-hydroxydopamine polymer, and epinephrine polymer particles.
  • a PDMS film containing the fluorescent nanoparticles can be provided.
  • it can be manufactured as a film-type composite (PFNPs-PDMS) in which dopamine and/or its analog nanoparticles are uniformly supported on polydimethylsiloxane (PDMS), and such PFNPs-PDMS film has bioadhesive properties. This is excellent, and it is possible to monitor various in vivo phenomena through fluorescence imaging after insertion into the body.
  • PFNPs-PDMS film-type composite
  • PDMS polydimethylsiloxane
  • the imaging composition of the present invention may be in the form of a PDMS film containing the fluorescent nanoparticles.
  • the film is immersed in a Tris-HCl (pH 8.5) buffer solution in which fluorescent nanoparticles are dissolved at 1 to 100 g/mL, for example 5 to 20 g/mL, and incubated for about 1 hour to 48 hours, for example. After 2 to 4 hours, the produced PFNPs-PDMS can be washed and dried.
  • the washing method is not particularly limited, and may be washed several times using, for example, distilled water. Additionally, the drying method is not particularly limited and may be performed, for example, by natural drying, freeze drying, hot air drying, oven drying, etc.
  • a hydrogel containing the fluorescent nanoparticles of the present invention, PVA, and water is provided.
  • the hydrogel may be an injectable hydrogel.
  • the hydrogel is prepared by mixing 0.5 to 5 g, for example, 1 to 3 g of PVA and 100 to 500 mg, for example, 150 to 350 mg, of fluorescent nanoparticles in 100 mL of water at 80 to 99°C, e.g.
  • PFNPs-PVA hydrogel can be prepared by heating at a temperature of 85 to 95°C for 30 minutes to 12 hours, for example, 1 hour to 4 hours, followed by gelation and cooling at room temperature.
  • the fluorescent nanoparticles of the present invention can be used for photothermal therapy.
  • the hydrogel as described above can be used for photothermal therapy.
  • the PFNPs-PVA hydrogel of the present invention has an excellent photothermal conversion effect of absorbing light at a near-infrared wavelength and emitting heat. Therefore, for example, by selectively injecting into cancer cell areas and then irradiating light at a near-infrared wavelength, photothermal conversion is achieved. The generation of heat through conversion can suppress the activity of cancer cells, resulting in an efficient treatment effect.
  • the cancer to which the photothermal treatment of the present invention can be applied may be classified as solid cancer or include cancer that causes metastasis of cancer cells, preferably breast cancer, melanoma, brain cancer, lung cancer, stomach cancer, liver cancer, head cancer, and uterus. This may include cervical cancer, prostate cancer, pancreatic cancer, colon cancer, and lymphoma.
  • the dopamine analogues used in the present invention levodopamine, norpinephrine, epinephrine, 6-hydroxydopamine, and dopamine, were mixed with 100 mg and 5 mL of ethylenediamine, respectively.
  • ethylenediamine was added to an alkaline solution mixed with 20 mL of distilled water, 9 mL of ethanol, and 1 mL of ammonia (NH 4 OH), and then reacted at room temperature for 1, 5, and 8 hours, respectively, resulting in polydopamine-based fluorescent nanoparticles that exhibit strong fluorescence.
  • PFNPs Polydopamine-based Fluorescent Nanoparticles, PFNPs
  • each of the fluorescent nanoparticles levodopa (LVD), nornephpyrine (NPP), 6-hydroxydopamine (HDA), dopamine (DA), and epinephrine (EPP), which are fluorescent nanoparticles prepared from dopamine analogues ( a) TEM (Transmission electron microscope), (b) XPS (X-ray photoelectron spectroscopy) spectrum, (c) FT-IR (Fourier transform infrared) spectrum, and (d) Zeta-potential analysis were performed. .
  • TEM Transmission electron microscope
  • XPS X-ray photoelectron spectroscopy
  • FT-IR Fastier transform infrared
  • the elemental compositions of LVD, NPP, HDA, DA, and EPP fNPs were analyzed from energy dispersive X-ray spectroscopy (EDS) analysis using a scanning electron microscope (SEM).
  • EDS energy dispersive X-ray spectroscopy
  • XPS X-ray photoelectron spectroscope
  • XPS spectra were observed to analyze the binding energy of each fluorescent nanoparticle prepared from dopamine and its analogs (a) LVD, (b) NPP, (c) HDA, (d) DA, and (e) EPP. .
  • C ketones
  • C1s carbon
  • N1s nitrogen
  • O1s oxygen
  • Polydopamine-based fluorescent nanoparticles prepared from dopamine and its analogues of the present invention become fluorescent through chemical degradation from nucleophiles such as ethylenediamine (EDA) as polymerization progresses in basic aqueous solutions. This occurred. Additionally, during the polymerization process, bandgap energy hybridization occurs due to structural differences in monomers, which generates monomer-dependent fluorescence wavelength diversity.
  • EDA ethylenediamine
  • polydopamine-based fluorescent nanoparticles of the present invention have various band gap hybridizations due to structural differences in the monomers from which they are produced. Therefore, polydopamine-based fluorescent nanoparticles prepared from various dopamine analogues have a range from at least 450 nm. Fluorescence was observed to be emitted at a wavelength of up to 650 nm or more ( Figure 4).
  • Polydopamine-based fluorescent nanoparticles prepared from dopamine analogues of the present invention generate various band gaps due to band gap hybridization and exhibit different fluorescence lifetimes accordingly.
  • PFNPs Polydopamine-based fluorescent nanoparticles prepared from different dopamine analogues of the present invention enable bio-imaging in animal cells and animals due to low toxicity and stability and diversity of fluorescence, and can provide bio-imaging and monitoring technology through this. there is.
  • MRC-5 cells were treated with polydopamine-based fluorescent nanoparticles prepared from dopamine and its analogs. 24 hours later, fluorescence images of the cells were observed using DAPI, GFP, and RFP filters of a fluorescence microscope. As a result, LVD, NPP, Multiple emission of blue, green, and red fluorescence due to HDA, DA, and EPP fNPs fluorescence was observed within MRC-5 cells ( Figure 6).
  • Levodopa (LVD) fluorescent nanoparticles are blue, nornephpyrine (NPP) fluorescent nanoparticles are green, 6-hydroxydopamine is yellow to light green, dopamine is orange, and epinephrine.
  • LLD Levodopa
  • NPP nornephpyrine
  • 6-hydroxydopamine is yellow to light green
  • dopamine is orange
  • epinephrine epinephrine
  • PFNPs based on polydopamine fluorescent nanoparticles of the present invention were tested for cytotoxicity using MRC-5 cells.
  • MRC-5 cells cultured in a 96-well plate at a concentration of 5.0x10 3 were treated with each PFNPs at a concentration of 50, 100, 200, 300, 400, and 500 ug/mL.
  • the PFNPs-treated supernatant was removed and treated with a 100uL volume of CCK-8 kit solution for 30 minutes.
  • the 450nm absorption wavelength of the processed 96-well plate was measured using a micro reader equipment.
  • PFNPs based on polydopamine fluorescent nanoparticles prepared from dopamine analogues show a cell survival rate of more than 85% for MRC-5 in the concentration range of 100 to 500ug/mL, showing very low toxicity. It was confirmed to have excellent biocompatibility.
  • bioadhesion is a field that can be applied in a variety of ways, such as biofilms, biopatches, and smart patches.
  • biofilms such as biofilms, biopatches, and smart patches.
  • mussels are a representative research target.
  • the fluorescent nanoparticles based on polydopamine, a similar mussel adhesive protein of the present invention contain a number of catechols and amines with bioadhesive properties on the surface.
  • PFNPs-PDMS film polydopamine-based fluorescent film
  • the bioadhesion ability of the PFNPs-PDMS film of the present invention was evaluated through a tack separation experiment.
  • An ABS type mold produced for tack separation evaluation was manufactured using a 3D printer, and in order to deposit a PFNPs-PDMS film on the manufactured mold, the tack was separated through taping, PDMS curing, and PFNPs doping. Separation) samples were produced for evaluation.
  • Figure 10 schematically shows the mold manufacturing process for testing the bioadhesion strength of this PFNPs-PDMS film.
  • a tack separation evaluation was conducted using pig skin as a biomaterial.
  • the ABS mold with the pig skin attached and the ABS mold with the PFNPs-PDMS film deposited on it are attached at room temperature while facing each other, and then a UTM (universal testing machine) is used through the process shown in FIG. 11. The evaluation was carried out.
  • the PFNPs-PDMS film manufactured using dopamine or its analogue LVD, HDA, NPP, DA, and EPP nanoparticles of the present invention is similar to the previously reported polydopamine-PDMS. It was confirmed that it exhibits 2 to 3 times better bioadhesion compared to film.
  • PFNPs-PDMS film of the present invention not only has excellent bioadhesiveness but also enables monitoring through in vivo fluorescence. Therefore, PFNPs-PDMS film was implanted into the rat body and in vivo fluorescence monitoring was performed. Specifically, PFNPs-PDMS films (0.5x0.5cm) prepared from LVD, HDA, NPP, DA, and EPP nanoparticles were implanted into the epidermis of 5-week-old balb/c nude mice. After 24 hours, fluorescence images were observed through Alexa514 and Cy3 filters using an in vivo imaging system (IVIS).
  • IVIS in vivo imaging system
  • the DA-PDMS film and EPP-PDMS film were implanted into the epidermis of a balb/c nude mouse and fluorescence imaging was observed for 15 days, resulting in deformation and fluorescence of the implantation site. It was confirmed that each film produced without a decrease in was capable of long-term fluorescence imaging in the biological area.
  • the epidermis of balb/c nude mice implanted with the PFNPs-PDMS film of the present invention was incised and histological toxicity analysis of the PFNPs-PDMS film was performed. Specifically, the epidermis of mice implanted with the PFNPs-PDMS film was fixed in formaldehyde and then assessed for toxicity to the epidermal tissue through H&E (hematoxylin and eosin staining), as can be seen in Figure 14. Abnormalities of the nucleus and cytosol were not observed in all epidermal tissues, indicating very low histological toxicity.
  • Photothermal therapy is a treatment that kills cancer cells by locally delivering materials such as nanoparticles to the area where cancer occurs and then irradiating a laser to generate heat at a certain temperature. It reduces the pain and side effects caused by existing cancer treatments such as surgery or chemical therapy. It is a new non-invasive cancer treatment that can be used.
  • the fluorescent nanoparticles of the present invention exhibit a photothermal conversion effect by near-infrared rays when combined with a polymer such as poly(vinylalcohol) (PVA), thereby producing an injectable hydrogel capable of treating cancer by killing cancer cells. did.
  • Figure 15 shows the hydrogel production and photothermal effect mechanism using the fluorescent nanoparticles of the present invention.
  • PFNPs-PVA hydrogels prepared from PVA and PFNPs were placed in a 1.5 mL transparent glass container, then irradiated with a 1W near-infrared (808 nm) laser for 10 minutes, and temperature changes were observed using a near-infrared camera.
  • FIG. 16 shows the time of each PFNPs-PVA hydrogel prepared from (a) PVA-hydrogel and dopamine analog (b) LVD, (c) NPP, (d) HDA, (e) DA, and (f) EPP. This shows the light-to-heat conversion effect.
  • Figure 17 shows the in vivo photothermal conversion effect of PFNPs-PVA hydrogel prepared from dopamine analogues LVD, NPP, HDA, DA, and EPP.
  • Photothermal treatment was performed by irradiating near-infrared rays every two days for 14 days after intratumoral injection of PFNPs-PVA hydrogel composite material into 6-week-old balb/c nude mice in which 4T1 cancer cells were grown to a size of 80 mm 3 in a volume of 100 uL. The effect of suppressing the activity of cancer cells was confirmed.
  • Figure 18 shows (a) temperature change in mice injected with PFNPs-PVA hydrogel over time according to near-infrared laser irradiation, (b) body weight change, and (c) change in cancer cell volume of mice following photothermal treatment for 14 days.
  • the temperature of mice irradiated with near-infrared PFNPs-hydrogels (LVD-PVA, NPP-PVA, HDA-PVA, DA-PVA, EPP-PVA) was higher within 5 minutes.
  • no change in the rat's body weight was observed during 14 days of photothermal treatment, and the volume of cancer cells decreased by up to 6 times or more.
  • the cancer treatment effect of PFNPs-hydrogel showed slightly different effects depending on the type of dopamine analogue, and the treatment effect tended to increase as the temperature increased.
  • Figure 19 shows the results of H&E staining of cancer cells after photothermal treatment using PFNPs-PVA hydrogel.
  • abnormality of the nucleus and cytosol in the cancer cell tissue of mice (control) injected with PBS was observed.
  • traces of cell death, such as shrinkage of the nucleus and cytoplasm due to photothermal treatment, were found in cancer cell tissue that underwent photothermal treatment after injection of PFNPs-PVA hydrogel.
  • tissue cells of the major organs of balb/c nude mice (heart, lung, liver, spleen, and kidney) were excised and treated with formaldehyde. ) and then analyzed by H&E (hematoxylin and eosin) staining.
  • Figure 20 shows the H&E staining results of heart, lung, liver, spleen, and kidney cells after photothermal treatment using PFNPs-PVA hydrogel.
  • PFNPs-PVA like the major organ tissues of mice (control) injected with PBS, Abnormalities of the nucleus and cytosol were not observed in the major organ tissues of mice that underwent photothermal treatment after injection of the hydrogel.
  • photothermal treatment using PFNPs-PVA hydrogel was effective in the heart, lungs, and liver. It was confirmed that it was not toxic to spleen and kidney tissues and showed a specific inhibitory effect on cancer cells.

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Abstract

The present invention relates to fluorescent nanoparticles, a preparation method therefor and a use thereof, and provided are: a preparation method for fluorescent nanoparticles, comprising a step of mixing dopamine or dopamine analogs with C1-C5alkylenediamine in a basic solvent; fluorescent nanoparticles which are polymers of dopamine or dopamine analogs; an imaging composition which comprises the fluorescent nanoparticles of the present invention and can image a plurality of areas; and a hydrogel comprising the fluorescent nanoparticles of the present invention, PVA and water.

Description

도파민 또는 도파민 유사체를 이용한 다기능성 바이오메디컬 형광 나노입자, 이의 제조방법 및 이의 용도Multifunctional biomedical fluorescent nanoparticles using dopamine or dopamine analogues, method of manufacturing the same, and use thereof
본 발명은 도파민 또는 도파민 유사체를 이용한 다기능성 바이오메디컬 형광 나노입자, 이의 제조방법 및 이의 용도에 관한 것으로, 보다 상세하게는 도파민 및 이의 유사체의 중합체로 이루어진 형광 나노입자, 이의 제조방법 및 이를 이미징, 광열치료 등에 적용하기 위한 다양한 용도에 관한 것이다.The present invention relates to multifunctional biomedical fluorescent nanoparticles using dopamine or dopamine analogues, a method of manufacturing the same, and uses thereof. More specifically, fluorescent nanoparticles made of a polymer of dopamine and its analogues, a method of manufacturing the same, and imaging the same. It relates to various uses for application to photothermal therapy, etc.
최근 수년에 걸쳐 생체의 생물학적 환경과 접촉하도록 고안된 의학적 및 약제학적 분야에서 신규한 재료의 적용에 대한 관심이 증가하고 있다. 이들 재료 중에서 중합체, 주로 합성 중합체가 지금까지는 환자의 건강 보호에 상당한 이점을 제공하는 것으로 밝혀진 가장 광범위한 종류이다.In recent years there has been increasing interest in the application of novel materials in medical and pharmaceutical fields designed to come into contact with the biological environment of living organisms. Among these materials, polymers, mainly synthetic polymers, are by far the most widespread class that has been shown to offer significant benefits in protecting patient health.
의학적 분야에 있어서 중합체의 용도는 임플란트 또는 인공 기관, 인공 혈관, 안내 렌즈, 인공 관절, 인공 보철, 봉합 재료, 체외 요법재 등과 같은 지지 재료 또는 혈액 관류, 혈액 산소 공급기, 카테터(catheter), 혈액 튜빙(tubing), 창상 및 화상 커버링 재료, 부목, 콘택트 렌즈 등에 사용되는 것과 같은 기타 지지 재료로서 사용된다. 약제학적 분야에 있어서, 중합체는 나노 입자 전달 시스템 및 방출 조절 전달 시스템의 개발에 사용되어, 목적하는 위치에 약물 전달을 표적화 하는 연구가 이루어져 왔다. 또한, 중합체는 경피 약물 전달 패치(patch), 미소구, 효소 및 세포 고정 등과 같은 응용 생물학적 요법 등에도 사용하는 것으로 밝혀졌다.Uses of polymers in the medical field include support materials such as implants or artificial organs, artificial blood vessels, intraocular lenses, artificial joints, prosthetics, suture materials, extracorporeal therapy materials, etc., or blood perfusion, blood oxygenators, catheters, blood tubing. Other support materials such as those used in tubing, wound and burn covering materials, splints, contact lenses, etc. In the pharmaceutical field, polymers have been used in the development of nanoparticle delivery systems and controlled-release delivery systems, and research has been conducted to target drug delivery to a desired location. Additionally, polymers have been found to be used in applied biological therapies such as transdermal drug delivery patches, microspheres, enzymes, and cell immobilization.
도파민은 신경세포간에 메신저 역할을 하는 일종의 신경전달 물질(Neuro-transmitter)로서 카테콜(Catechol) 또는 카테킨(Catechin)의 화학적 성분을 포함한 아민(Catecholamines)의 일종이다. 이러한 도파민(DA)을 중합시키면 폴리도파민(Polydopamine: PDA)이 생성되며, 폴리도파민이 코팅된 표면은 아민(-NH2) 또는 티올(thiol)기를 가진 분자들을 공유결합으로 흡착시킴으로써 다양한 작용기를 도입할 수 있고, 특히 각종 금속과 산화규소, 산화철, 스테인리스, 테플론, 폴리스티렌 등 다양한 지지표면에 코팅시킬 수 있으며, 접착력이 매우 우수하다.Dopamine is a type of neurotransmitter that acts as a messenger between nerve cells and is a type of amine containing the chemical components of catechol or catechin. Polydopamine (PDA) is produced when dopamine (DA) is polymerized, and the polydopamine-coated surface introduces various functional groups by covalently adsorbing molecules with amine (-NH 2 ) or thiol groups. In particular, it can be coated on various supporting surfaces such as various metals, silicon oxide, iron oxide, stainless steel, Teflon, and polystyrene, and has excellent adhesion.
폴리도파민을 이용한 기술로는 한국등록특허 제10-1539389 B1호가 폴리도파민 유도 플라즈모닉 나노하이브리드를 이용한 보조인자 재생방법을 개시하고 있으며, 기판에 폴리도파민, 금속 나노입자 및 염료를 첨가하여 제조된 폴리도파민 유도 플라즈모닉 나노하이브리드를 이용하여 보조인자를 재생하고, 상기 재생된 보조인자를 사용하여 산화환원효소 반응으로 유용물질을 제조하는 인공광합성 방법을 개시하고 있다. As a technology using polydopamine, Korean Patent No. 10-1539389 B1 discloses a cofactor regeneration method using polydopamine-derived plasmonic nanohybrid, and polydopamine produced by adding polydopamine, metal nanoparticles, and dye to the substrate An artificial photosynthesis method is disclosed in which cofactors are regenerated using a dopamine-induced plasmonic nanohybrid and useful substances are produced through an oxidoreductase reaction using the regenerated cofactors.
나아가, 도파민(dopamine)을 포함하는 도파민 유사체로부터 형광 소재로서 바이오메디컬 분야에 응용 가능한 다기능성 복합 나노입자를 제조하는 기술 및 이렇게 제조된 형광 나노입자가 제공되는 경우 관련 분야에서 널리 적용될 수 있을 것으로 기대된다.Furthermore, if the technology for manufacturing multifunctional composite nanoparticles that can be applied in the biomedical field as a fluorescent material from dopamine analogs containing dopamine and the fluorescent nanoparticles prepared in this way are provided, it is expected that they can be widely applied in related fields. do.
이에 본 발명의 한 측면은 형광 나노입자의 제조방법을 제공하는 것이다.Accordingly, one aspect of the present invention is to provide a method for producing fluorescent nanoparticles.
본 발명의 다른 측면은 생체 적용성이 우수한 형광 나노입자를 제공하는 것이다.Another aspect of the present invention is to provide fluorescent nanoparticles with excellent bioapplicability.
본 발명의 또 다른 측면은 형광 나노입자를 포함하는 이미징 조성물을 제공하는 것이다.Another aspect of the present invention is to provide an imaging composition comprising fluorescent nanoparticles.
본 발명의 또 다른 측면은 형광 나노입자를 포함하는 하이드로겔을 제공하는 것이다.Another aspect of the present invention is to provide a hydrogel containing fluorescent nanoparticles.
본 발명의 일 견지에 의하면, 도파민(dopamine) 또는 도파민 유사체를 염기성 용매 하에서 C1-C5알킬렌디아민과 혼합하는 단계를 포함하는, 형광 나노입자의 제조방법이 제공된다.According to one aspect of the present invention, a method for producing fluorescent nanoparticles is provided, comprising mixing dopamine or a dopamine analog with C 1 -C 5 alkylenediamine in a basic solvent.
본 발명의 다른 견지에 의하면, 도파민(dopamine) 또는 도파민 유사체의 중합체인, 형광 나노입자가 제공된다.According to another aspect of the present invention, fluorescent nanoparticles that are polymers of dopamine or a dopamine analog are provided.
본 발명의 또 다른 견지에 의하면, 본 발명의 형광 나노입자를 포함하여 복수 영역의 이미징이 가능한, 이미징 조성물이 제공된다.According to another aspect of the present invention, an imaging composition capable of imaging multiple areas including the fluorescent nanoparticles of the present invention is provided.
본 발명의 또 다른 견지에 의하면, 본 발명의 형광 나노입자, PVA 및 물을 포함하는, 하이드로겔이 제공된다.According to another aspect of the present invention, a hydrogel is provided, comprising the fluorescent nanoparticles of the present invention, PVA, and water.
본 발명의 형광 나노입자는 그 제조 공정이 간단하고, 이를 이용한 생체 접착 필름 및 하이드로겔로 구현되는 경우, 이는 생체 조직에 대한 우수한 접착성으로 생체 고정화 및 형광을 통한 모니터링에 높은 활용성이 있을 뿐만 아니라, 근적외선 파장의 조사(irradiation)에 따른 광열 전환 효율이 높아 광열 치료, 예를 들어 암세포 특이적인 광열 치료에도 적용이 가능하다.The fluorescent nanoparticles of the present invention have a simple manufacturing process, and when implemented as bioadhesive films and hydrogels using them, they not only have excellent adhesion to biological tissues, but are highly useful for bioimmobilization and monitoring through fluorescence. In addition, the photothermal conversion efficiency due to irradiation of near-infrared wavelengths is high, so it can be applied to photothermal therapy, for example, cancer cell-specific photothermal treatment.
도 1은 도파민 및 도파민 유사체를 이용한 형광 나노입자(PFNPs) 제조 공정을 도식적으로 나타낸 것이다.Figure 1 schematically shows the manufacturing process of fluorescent nanoparticles (PFNPs) using dopamine and dopamine analogues.
도 2는 레보도파(LVD), 노르네프피린(NPP), 6-하이드록시도파민(HDA), 도파민(DA), 에피네프린(EPP)으로부터 제조된 PFNPs의 (a) TEM, (b) XPS 스펙트럼, (c) FT-IR 스펙트럼, (d) Zeta potential 분석 결과를 나타낸 것이다.Figure 2 shows (a) TEM, (b) c) FT-IR spectrum, (d) Zeta potential analysis results.
도 3은 도파민 및 도파민 유사체로부터 합성된 PFNPs의 X선 광전자 분광법(XPS narrow) 스펙트럼 분석 결과를 나타낸 것이다. Figure 3 shows the results of X-ray photoelectron spectroscopy (XPS narrow) spectrum analysis of PFNPs synthesized from dopamine and dopamine analogues.
도 4는 본 발명의 형광 나노입자(PFNPs)의 구조적 차이에 따른 방출 형광스펙트럼을 나타낸 것이다.Figure 4 shows the emission fluorescence spectrum of the fluorescent nanoparticles (PFNPs) of the present invention according to structural differences.
도 5는 본 발명의 형광 나노입자(PFNPs)의 (a) 형광수명(fluorescence lifetime) 스펙트럼 및 (b) 고분자화 반응시간에 따른 형광 스펙트럼 변화를 나타낸 것이다.Figure 5 shows (a) the fluorescence lifetime spectrum of the fluorescent nanoparticles (PFNPs) of the present invention and (b) the change in fluorescence spectrum according to the polymerization reaction time.
도 6은 본 발명의 폴리도파민 기반 형광 나노입자(LVD, NPP, HDA, DA, EPP fNPs)의 MRC-5 세포 내 형광 이미지를 나타낸 것이다. Figure 6 shows fluorescence images of the polydopamine-based fluorescent nanoparticles (LVD, NPP, HDA, DA, EPP fNPs) of the present invention in MRC-5 cells.
도 7은 본 발명의 폴리도파민 기반 형광 나노입자의 생체 내 형광 이미지를 나타낸 것이다. 레보도파(LVD) 형광 나노입자는 푸른색, 노르네프피린(NPP) 형광 나노입자는 녹색, 6-하이드록시도파민(6-hydroxydopamine)은 노란색 내지 연두색, 도파민(dopamine)은 주황색, 그리고 에피네프린(epinephrine)은 붉은색의 형광 이미지를 각각 나타낼 수 있다.Figure 7 shows an in vivo fluorescence image of the polydopamine-based fluorescent nanoparticles of the present invention. Levodopa (LVD) fluorescent nanoparticles are blue, nornephpyrine (NPP) fluorescent nanoparticles are green, 6-hydroxydopamine is yellow to light green, dopamine is orange, and epinephrine. Each can display a red fluorescence image.
도 8은 본 발명의 폴리도파민 형광 나노입자 기반 (a) LVD, (b) NPP, (c) HDA, (d) DA, (e) EPP fNPs의 MRC-5 세포에 대한 세포 독성 결과를 나타낸 것이다. Figure 8 shows the cytotoxicity results of (a) LVD, (b) NPP, (c) HDA, (d) DA, and (e) EPP fNPs based on polydopamine fluorescent nanoparticles of the present invention on MRC-5 cells. .
도 9 (a)는 PFNPs-PDMS 필름의 제조 공적을 도식적으로 나타낸 것이고, 도 9 (b)는 제조된 PFNPs-PDMS 필름의 이미지를 나타낸 것이다.Figure 9 (a) schematically shows the manufacturing process of the PFNPs-PDMS film, and Figure 9 (b) shows an image of the prepared PFNPs-PDMS film.
도 10은 PFNPs-PDMS 필름의 생체 접착강도 실험을 위한 몰드 제작 과정을 도식적으로 나타낸 것이다.Figure 10 schematically shows the mold manufacturing process for testing the bioadhesion strength of the PFNPs-PDMS film.
도 11은 PFNPs-PDMS 필름의 택 분리(Tack separation) 실험의 평가 방법을 도식적으로 나타낸 것이다.Figure 11 schematically shows the evaluation method of the tack separation experiment of the PFNPs-PDMS film.
도 12 (a)는 UTM을 이용한 접착 에너지 계산 방법을 나타낸 것이고, 도 12 (b) 및 (c)는 UTM으로부터 측정된 PFNP-PDMS 필름의 접착 강도를 비교한 결과를 나타낸 것이다. Figure 12 (a) shows a method for calculating adhesion energy using UTM, and Figures 12 (b) and (c) show the results of comparing the adhesion strength of the PFNP-PDMS film measured from UTM.
도 13은 PFNPs-PDMS가 삽입된 쥐의 형광 이미지를 나타낸 것이다. Figure 13 shows a fluorescence image of a mouse implanted with PFNPs-PDMS.
도 14는 H&E 염색을 통한 PFNPs-PDMS 필름의 조직학적 독성 분석 결과를 나타낸 것이다.Figure 14 shows the results of histological toxicity analysis of the PFNPs-PDMS film through H&E staining.
도 15는 본 발명의 형광 나노입자를 이용한 하이드로겔 제작 및 광열 효과 메커니즘을 도식적으로 나타낸 것이다. Figure 15 schematically shows the hydrogel production and photothermal effect mechanism using the fluorescent nanoparticles of the present invention.
도 16은 (a) PVA-하이드로겔 및 도파민 유사체 (b) LVD, (c) NPP, (d) HDA, (e) DA, (f) EPP로부터 제조된 PFNPs-PVA 하이드로겔의 시간에 따른 광열 변환 효과를 나타낸 것이다. Figure 16 shows photothermal properties of PFNPs-PVA hydrogel prepared from (a) PVA-hydrogel and dopamine analog (b) LVD, (c) NPP, (d) HDA, (e) DA, and (f) EPP. This shows the conversion effect.
도 17은 본 발명의 형광 나노입자를 이용한 하이드로겔의 생체 내 광열 변환 효과를 나타낸 것이다.Figure 17 shows the in vivo photothermal conversion effect of a hydrogel using the fluorescent nanoparticles of the present invention.
도 18은 (a) 근적외선 레이져 조사에 따른 시간별 PFNPs-PVA 하이드로겔을 주입한 쥐의 온도변화, (b) 14일 동안의 광열 치료에 따른 쥐의 체중 변화 및 (c) 암세포 부피의 변화를 나타낸 것이다.Figure 18 shows (a) temperature change of mice injected with PFNPs-PVA hydrogel over time according to near-infrared laser irradiation, (b) change in body weight of mice following photothermal treatment for 14 days, and (c) change in cancer cell volume. will be.
도 19는 PFNPs-PVA 하이드로겔을 이용한 광열 치료 후 암 세포의 H&E 염색(staining) 결과를 나타낸 것이다.Figure 19 shows the H&E staining results of cancer cells after photothermal treatment using PFNPs-PVA hydrogel.
도 20은 PFNPs-PVA 하이드로겔을 이용한 광열 치료 후 심장, 폐, 간, 지라, 신장 세포의 H&E 염색(staining) 결과를 나타낸 것이다.Figure 20 shows the H&E staining results of heart, lung, liver, spleen, and kidney cells after photothermal treatment using PFNPs-PVA hydrogel.
도 21은 DA-PDMS 필름 및 EPP-PDMS 필름을 balb/c 누드(nude) 마우스 표피에 생체 표피 조직에 이식하여 형광 이미징을 15일 동안 관측한 결과를 나타낸 것으로, (a) balb/c nude 쥐의 fNPs-PDMS 이식 전 후 사진, (b) DA fNPs PDMS 및 EPP fNPs PDMS 필름이 주입 된 쥐의 생체 형광 이미징 (Cy 3 파장 영역), 및 (c) DA fNPs PDMS 및 EPP fNPs PDMS 필름이 주입 된 쥐의 정량적 형광 세기 변화 그래프(15일, 3일 간격)를 나타낸 것이다.Figure 21 shows the results of fluorescence imaging observed for 15 days by transplanting the DA-PDMS film and EPP-PDMS film into the epidermis of a balb/c nude mouse and living epidermal tissue, (a) balb/c nude mouse Photographs before and after fNPs-PDMS implantation, (b) biofluorescence imaging (Cy 3 wavelength region) of mice injected with DA fNPs PDMS and EPP fNPs PDMS films, and (c) mice injected with DA fNPs PDMS and EPP fNPs PDMS films. This shows a graph of quantitative fluorescence intensity change in mice (15 days, 3-day intervals).
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. Hereinafter, preferred embodiments of the present invention will be described with reference to the attached drawings. However, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명에 의하면 도파민 및/또는 이의 유사체를 기반으로 하는 형광 나노입자의 제조 방법이 제공된다. According to the present invention, a method for producing fluorescent nanoparticles based on dopamine and/or its analogs is provided.
본 발명에 의해 제공되는 형광 나노입자는 PFNPs(Polydopamine-based Fluorescent Nanoparticles) 또는 fNPs로 상호호환적으로 지칭될 수 있다.The fluorescent nanoparticles provided by the present invention may be referred to interchangeably as PFNPs (Polydopamine-based Fluorescent Nanoparticles) or fNPs.
본 발명의 형광 나노입자의 제조방법은 도파민(dopamine) 또는 도파민 유사체를 염기성 용매 하에서 C1-C5알킬렌디아민과 혼합하는 단계를 포함하는 것이다. The method for producing fluorescent nanoparticles of the present invention includes the step of mixing dopamine or a dopamine analog with C 1 -C 5 alkylenediamine in a basic solvent.
이때, 상기 도파민 유사체는 레보도파민(levodopamine), 노르에피네프린(norepinephrine), 에피네프린(epinephrine), 및 6-하이드록시도파민(6-hydroxydopamine)으로 이루어진 그룹으로부터 선택되는 적어도 하나인 것일 수 있다.At this time, the dopamine analog may be at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine.
한편, 상기 C1-C5알킬렌디아민은 메틸렌디아님, 에틸렌디아민, 프로필렌디아민 등을 포함하는 것으로, 바람직하게는 에틸렌디아민인 것이다. Meanwhile, the C 1 -C 5 alkylenediamine includes methylenedianim, ethylenediamine, propylenediamine, etc., and is preferably ethylenediamine.
보다 상세하게, 본 발명의 형광 나노입자의 제조방법은 도파민 및 이의 유사체 중 적어도 하나의 성분을 C1-C5알킬렌디아민 및 암모니아를 포함하는 수용액에 혼합하여 상온에서 1시간 내지 24시간, 예를 들어 1시간 내지 12시간 동안 반응시켜 제조될 수 있다. 상기 수용액은 추가로 에탄올 등의 알코올을 포함할 수 있다. More specifically, the method for producing fluorescent nanoparticles of the present invention is to mix at least one component of dopamine and its analogs in an aqueous solution containing C 1 -C 5 alkylenediamine and ammonia for 1 to 24 hours at room temperature, e.g. For example, it can be prepared by reacting for 1 to 12 hours. The aqueous solution may additionally contain alcohol such as ethanol.
한편, 상기 염기성 용매는 암모니아 수용액인 것일 수 있으며, 암모니아는 상기 수용액 내에 2 내지 3%의 농로로 사용되는 것이 바람직하며, 암모니아의 함량이 상기 범위 미만인 경우에는 자가 조립을 통한 중합의 어려움이 발생할 수 있다. Meanwhile, the basic solvent may be an ammonia aqueous solution, and ammonia is preferably used in an amount of 2 to 3% in the aqueous solution. If the ammonia content is less than the above range, polymerization through self-assembly may be difficult. there is.
예를 들어 상기 도파민 또는 이의 유사체 50 내지 200mg, 예를 들어 100 내지 150mg을 1 내지 10 mL, 예를 들어 3 내지 7mL의 C1-C5알킬렌디아민, 10mL 내지 50 mL, 예를 들어 20 내지 40 mL의 증류수, 5 내지 15 mL, 예를 들어 7 내지 11mL의 에탄올, 및 0.5 내지 5mL의 암모니아수, 예를 들어 2 내지 3%의 농도의 암모니아수(NH4OH)가 혼합된 알칼라인 수용액에 넣어준 후 1 내지 8 시간, 예를 들어 1 내지 5 시간 동안 상온에서 반응시킬 수 있다. 본 발명의 상기 알칼라인 수용액은 pH가 9 내지 11, 예를 들어 pH가 9.5 내지 10.5인 것이 바람직하다.For example, 50 to 200 mg, for example 100 to 150 mg, of the dopamine or its analogue may be mixed with 1 to 10 mL, for example 3 to 7 mL of C 1 -C 5 alkylenediamine, 10 mL to 50 mL, for example 20 to 7 mL. Added to an alkaline aqueous solution mixed with 40 mL of distilled water, 5 to 15 mL, for example, 7 to 11 mL of ethanol, and 0.5 to 5 mL of ammonia water, for example, ammonia water (NH 4 OH) with a concentration of 2 to 3%. Afterwards, the reaction can be carried out at room temperature for 1 to 8 hours, for example, 1 to 5 hours. The alkaline aqueous solution of the present invention preferably has a pH of 9 to 11, for example, 9.5 to 10.5.
예를 들어, 상기 C1-C5알킬렌디아민은 도파민 및/또는 이의 유사체가 용해되어 있는 30mL의 (수)용액에 대하여 0.5mL 내지 5 mL의 부피비로 사용하는 것이 바람직하며, C1-C5알킬렌디아민이 상기 범위 미만인 경우에는 수율이 낮아지는 문제가 있고, 상기 범위를 초과하는 경우에는 고분자의 중량평균 분자량이 낮아질 수 있다.For example, the C 1 -C 5 alkylenediamine is preferably used in a volume ratio of 0.5 mL to 5 mL with respect to 30 mL of (aqueous) solution in which dopamine and/or its analogs are dissolved, and C 1 -C 5 If the alkylenediamine is less than the above range, there is a problem of low yield, and if it exceeds the above range, the weight average molecular weight of the polymer may be low.
상기와 같은 본 발명의 형광 나노입자의 제조방법에 의하면 도파민(dopamine) 및/또는 도파민 유사체의 중합체인, 형광 나노입자가 제조될 수 있다. 본 발명의 형광 나노입자는 형광 소재로서 바이오메디컬 분야에 응용 가능한 다기능성 복합 나노입자이며, 낮은 독성 및 다색(Red, Green, Blue) 형광을 이용한 동물세포에 대한 다중 바이오이미징이 가능하다. According to the method for producing fluorescent nanoparticles of the present invention as described above, fluorescent nanoparticles, which are polymers of dopamine and/or dopamine analogs, can be produced. The fluorescent nanoparticles of the present invention are multifunctional composite nanoparticles that can be applied to the biomedical field as a fluorescent material, and enable multiple bio-imaging of animal cells using low toxicity and multicolor (red, green, blue) fluorescence.
본 발명의 상기 형광 나노입자는 50 내지 300nm의 입경의 크기를 갖는 나노입자일수 있다. 예를 들어 각 나노입자의 입경은 50 내지 200 nm이며 중합 시간 및 단량체의 첨가량에 따라 조절될 수 있다.The fluorescent nanoparticles of the present invention may be nanoparticles with a particle diameter of 50 to 300 nm. For example, the particle size of each nanoparticle is 50 to 200 nm and can be adjusted depending on the polymerization time and the amount of monomer added.
이때 도파민 유사체는 레보도파민(levodopamine), 노르에피네프린(norepinephrine), 에피네프린(epinephrine), 및 6-하이드록시도파민(6-hydroxydopamine)으로 이루어진 그룹으로부터 선택되는 적어도 하나인 것일 수 있으며, 예를 들어 상기 형광 나노입자는 레보도파민 폴리머, 6-하이드록시도파민 폴리머 및 에피네프린 폴리머 입자 중 적어도 하나를 포함하는 형광 나노입자의 조합인 것일 수 있다. At this time, the dopamine analog may be at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine, for example, the fluorescence The nanoparticles may be a combination of fluorescent nanoparticles containing at least one of levodopamine polymer, 6-hydroxydopamine polymer, and epinephrine polymer particles.
본 발명에 의하면 형광 나노입자를 포함하는 이미징 조성물이 제공되며, 예를 들어 복수 종의 형광 나노입자를 포함하여 복수 영역의 이미징이 가능한, 이미징 조성물이 제공될 수 있다. 이들 복수 종의 형광 나노입자는 상이한 분자 구조에 따라 빨강, 초록, 파랑 등의 각각 다른 파장의 형광 방출하는 특성을 나타내며, 이를 기초로 동물 세포에 대한 다중 형광 이미징이 가능하다. 예를 들어 상기 복수 종의 형광 나노입자는 레보도파민 폴리머, 6-하이드록시도파민 폴리머 및 에피네프린 폴리머 입자 중 적어도 하나를 포함하는 형광 나노입자의 조합인 것일 수 있다.According to the present invention, an imaging composition containing fluorescent nanoparticles is provided. For example, an imaging composition capable of imaging multiple areas including multiple types of fluorescent nanoparticles may be provided. These multiple types of fluorescent nanoparticles exhibit the characteristic of emitting fluorescence of different wavelengths such as red, green, and blue depending on their different molecular structures, and based on this, multiple fluorescence imaging of animal cells is possible. For example, the plurality of types of fluorescent nanoparticles may be a combination of fluorescent nanoparticles containing at least one of levodopamine polymer, 6-hydroxydopamine polymer, and epinephrine polymer particles.
본 발명에 의하면 상기 형광 나노입자를 포함하는 PDMS 필름이 제공될 수 있다. 보다 상세하게 폴리디메틸실록산(polydimethylsiloxane, PDMS)에 도파민 및/또는 이의 유사체 나노입자를 균일하게 담지한 필름 형태의 복합체(PFNPs-PDMS)로 제조가 가능하며, 이와 같은 PFNPs-PDMS 필름은 생체 접착성이 매우 우수하며 생체 내에 삽입 후 형광 이미징을 통하여 다양한 생체 내 현상에 대한 모니터링이 가능하다. According to the present invention, a PDMS film containing the fluorescent nanoparticles can be provided. In more detail, it can be manufactured as a film-type composite (PFNPs-PDMS) in which dopamine and/or its analog nanoparticles are uniformly supported on polydimethylsiloxane (PDMS), and such PFNPs-PDMS film has bioadhesive properties. This is excellent, and it is possible to monitor various in vivo phenomena through fluorescence imaging after insertion into the body.
이에, 본 발명의 상기 이미징 조성물은 상기 형광 나노입자를 포함하는 PDMS 필름의 형태인 것일 수 있다.Accordingly, the imaging composition of the present invention may be in the form of a PDMS film containing the fluorescent nanoparticles.
상기 필름은 예를 들어 형광 나노입자가 1 내지 100 g/mL, 예를 들어 5 내지 20 g/mL 용해되어 있는 Tris-HCl(pH 8.5) 완충 용액에 담그고 약 1시간 내지 48 시간, 예를 들어 2시간 내지 4시간이 지난 후, 제작된 PFNPs-PDMS를 세척하고 건조하여 획득될 수 있다. 이때 세척 방법은 특히 제한되는 것은 아니며, 예를 들어 증류수를 이용하여 수회 세척할 수 있다. 또한 건조 방식 역시 특히 제한되는 것은 아니며, 예를 들어 자연 건조, 동결건조, 열풍 건조, 오븐 건조 등에 의해 수행될 수 있다.The film is immersed in a Tris-HCl (pH 8.5) buffer solution in which fluorescent nanoparticles are dissolved at 1 to 100 g/mL, for example 5 to 20 g/mL, and incubated for about 1 hour to 48 hours, for example. After 2 to 4 hours, the produced PFNPs-PDMS can be washed and dried. At this time, the washing method is not particularly limited, and may be washed several times using, for example, distilled water. Additionally, the drying method is not particularly limited and may be performed, for example, by natural drying, freeze drying, hot air drying, oven drying, etc.
나아가, 본 발명에 의하면 본 발명의 형광 나노입자, PVA 및 물을 포함하는, 하이드로겔이 제공된다. 예를 들어 상기 하이드로겔은 주입용(injectable) 하이드로겔인 것일 수 있다. Furthermore, according to the present invention, a hydrogel containing the fluorescent nanoparticles of the present invention, PVA, and water is provided. For example, the hydrogel may be an injectable hydrogel.
예를 들어 상기 하이드로겔은 100 mL의 물에 0.5 내지 5g, 예를 들어 1 내지 3g의 PVA 및 형광 나노입자 100 내지 500 mg, 예를 들어 150 내지 350mg을 혼합한 혼합물을 80 내지 99℃, 예를 들어 85 내지 95℃의 온도에서 30분 내지 12 시간, 예를 들어 1 시간 내지 4시간 동안 가열한 후 겔레이션(gelation)하고 상온에서 냉각시켜 PFNPs-PVA 하이드로겔로 제조될 수 있다. For example, the hydrogel is prepared by mixing 0.5 to 5 g, for example, 1 to 3 g of PVA and 100 to 500 mg, for example, 150 to 350 mg, of fluorescent nanoparticles in 100 mL of water at 80 to 99°C, e.g. For example, PFNPs-PVA hydrogel can be prepared by heating at a temperature of 85 to 95°C for 30 minutes to 12 hours, for example, 1 hour to 4 hours, followed by gelation and cooling at room temperature.
본 발명의 형광 나노입자는 광열치료용도로 사용될 수 있으며, 예를 들어 상기와 같은 상기 하이드로겔은 광열치료용일 수 있다. 즉, 본 발명의 PFNPs-PVA 하이드로겔은 근적외선 파장의 빛을 흡수하여 열을 방출하는 광열 변환 효과가 우수하며, 따라서 예를 들어 암 세포부위에 대한 선택적인 주입 후 근적외선 파장의 빛을 조사함으로서 광열 변환을 통한 열의 발생이 암세포의 활성을 억제하여 이에 따른 효율적인 치료 효과를 도출할 수 있다. The fluorescent nanoparticles of the present invention can be used for photothermal therapy. For example, the hydrogel as described above can be used for photothermal therapy. In other words, the PFNPs-PVA hydrogel of the present invention has an excellent photothermal conversion effect of absorbing light at a near-infrared wavelength and emitting heat. Therefore, for example, by selectively injecting into cancer cell areas and then irradiating light at a near-infrared wavelength, photothermal conversion is achieved. The generation of heat through conversion can suppress the activity of cancer cells, resulting in an efficient treatment effect.
본 발명의 광열치료가 적용될 수 있는 상기 암은 고형암으로 분류되거나 암세포의 전이를 유발하는 암을 포함할 수 있으며, 바람직하게는, 유방암, 흑색종, 뇌암, 폐암, 위암, 간암, 두부암, 자궁경부암, 전립선암, 췌장암, 대장암, 림프종을 포함할 수 있다.The cancer to which the photothermal treatment of the present invention can be applied may be classified as solid cancer or include cancer that causes metastasis of cancer cells, preferably breast cancer, melanoma, brain cancer, lung cancer, stomach cancer, liver cancer, head cancer, and uterus. This may include cervical cancer, prostate cancer, pancreatic cancer, colon cancer, and lymphoma.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.
실시예Example
1. 본 발명의 형광 나노입자(PFNPs) 제조 방법1. Method for producing fluorescent nanoparticles (PFNPs) of the present invention
본 발명에서 사용된 도파민 유사체인 레보도파민(levodopamine), 노르피네프린(norpinephrine), 에피네프린(epinephrine), 6-하이드록시도파민(6-hydroxydopamine), 그리고 도파민(dopamine)을 각각 100mg과 5mL의 에틸렌디아민 (ethylenediamine)을 증류수 20mL, 에탄올 9mL 암모니아수(NH4OH) 1mL로 혼합된 알칼라인 용액에 넣어준 후 각각 1, 5, 8시간 동안 상온에서 반응시켜 최종적으로 강한 형광을 나타내는 폴리도파민 기반의 형광 나노입자(Polydopamine-based Fluorescent Nanoparticles, PFNPs)를 제조하였다. 제조한 각각의 PFNPs를 12000PRM으로 원심분리(centrifugation) 및 48시간의 삼투(dialysis) 과정을 거쳐 정제하여 얻었다(도 1).　 The dopamine analogues used in the present invention, levodopamine, norpinephrine, epinephrine, 6-hydroxydopamine, and dopamine, were mixed with 100 mg and 5 mL of ethylenediamine, respectively. (ethylenediamine) was added to an alkaline solution mixed with 20 mL of distilled water, 9 mL of ethanol, and 1 mL of ammonia (NH 4 OH), and then reacted at room temperature for 1, 5, and 8 hours, respectively, resulting in polydopamine-based fluorescent nanoparticles that exhibit strong fluorescence. (Polydopamine-based Fluorescent Nanoparticles, PFNPs) were prepared. Each of the prepared PFNPs was purified through centrifugation at 12000 PRM and dialysis for 48 hours (Figure 1).
2. 본 발명의 형광 나노입자(PFNPs)의 구조 평가 2. Structural evaluation of fluorescent nanoparticles (PFNPs) of the present invention
가. TEM, XPS 스펙트럼, FT-IR 스펙트럼, 제타 포텐셜(Zeta potential) 분석을 통한 PFNPs의 구조 분석 go. Structural analysis of PFNPs through TEM, XPS spectrum, FT-IR spectrum, and Zeta potential analysis
도파민 유사체로부터 제조된 형광 나노입자인 레보도파(LVD), 노르네프피린(NPP), 6-하이드록시도파민(HDA), 도파민(DA), 에피네프린(EPP) 각각의 나노입자의 구조를 분석하기 위하여 (a) TEM(Trasnsmission electron microscope), (b) XPS(X-ray photoelectron spectroscopy) 스펙트럼, (c) FT-IR(Fourier transform infrared) 스펙트럼, (d) 제타 포텐션(Zeta-potential) 분석을 수행하였다. TEM 분석 결과, 도파민 유사체로부터 제조된 PFNPs는 50 내지 200nm의 크기를 가지는 나노입자 형태임을 확인하였다. (b) XPS의 분석결과에서는 도파민 유사체로부터 제조된 모든 PFNPs는 무기물 성분은 전혀 관찰되지 않고 탄소(C1s), 질소(N1s), 산소(O1s)의 유기물로만 구성되어 있음을 확인하였다. (c) FT-IR 분석 결과, 도파민 유사체로부터 제조된 모든 PFNPs 나노입자에서 폴리도파민 고분자 구조의 특징인 카테콜(catechol) 그룹에 기인하는 하이드록시(-OH, 3300cm-1) 관능기, 케톤(ketone, 1500cm-1) 관능기의 흡광도가 확인되었다. (d) 나노입자 표면의 전하 특성을 관찰하기 위한 제타전위 측정 결과, 각각의 도파민 유사체로부터 제조된 형광 나노입자는 유사체 간의 구조적 차이에 의한 서로 다른 제타전위가 측정이 됨을 확인하였다.(도 2)To analyze the structure of each of the fluorescent nanoparticles levodopa (LVD), nornephpyrine (NPP), 6-hydroxydopamine (HDA), dopamine (DA), and epinephrine (EPP), which are fluorescent nanoparticles prepared from dopamine analogues ( a) TEM (Transmission electron microscope), (b) XPS (X-ray photoelectron spectroscopy) spectrum, (c) FT-IR (Fourier transform infrared) spectrum, and (d) Zeta-potential analysis were performed. . As a result of TEM analysis, it was confirmed that PFNPs prepared from dopamine analogues were in the form of nanoparticles with a size of 50 to 200 nm. (b) XPS analysis results confirmed that all PFNPs prepared from dopamine analogues were composed of only organic substances of carbon (C1s), nitrogen (N1s), and oxygen (O1s), with no inorganic components observed at all. (c) As a result of FT-IR analysis, all PFNPs nanoparticles prepared from dopamine analogs contained hydroxy (-OH, 3300 cm -1 ) functional group and ketone due to the catechol group, which is a characteristic of the polydopamine polymer structure. , 1500 cm -1 ), the absorbance of the functional group was confirmed. (d) As a result of zeta potential measurement to observe the charge characteristics of the nanoparticle surface, it was confirmed that fluorescent nanoparticles prepared from each dopamine analog had different zeta potentials due to structural differences between the analogs (Figure 2).
나. SEM(Scanning electron microscope)을 이용한 PFNPs의 원소 조성 분석me. Analysis of elemental composition of PFNPs using SEM (Scanning electron microscope)
SEM(scanning electron microscope)의 EDS(energy dispersive X-ray spectroscopy) 분석으로부터 LVD, NPP, HDA, DA, EPP fNPs의 원소 조성을 분석하였다. The elemental compositions of LVD, NPP, HDA, DA, and EPP fNPs were analyzed from energy dispersive X-ray spectroscopy (EDS) analysis using a scanning electron microscope (SEM).
SEM을 이용한 PFNPs의 원소 조성 분석Elemental composition analysis of PFNPs using SEM
시료(Sample)Sample C (%)C (%) N (%)N (%) O (%)O (%)
LVD fNPsLVD fNPs 66.9366.93 18.2818.28 14.7914.79
NPP fNPsNPP fNPs 63.6463.64 26.6426.64 9.729.72
HDA fNPsHDA fNPs 66.3866.38 24.2524.25 9.379.37
DA fNPsDA fNPs 73.2073.20 18.7218.72 8.088.08
EPP fNPsEPP fNPs 63.9863.98 26.9026.90 9.139.13
분석결과, 도파민 유사체로부터　합성된 LVD, NPP, HDA, DA, EPP fNPs는 탄소(C) 기반의 유기 나노입자로 구성되어 있음을 확인하였다.　　 As a result of the analysis, it was confirmed that LVD, NPP, HDA, DA, and EPP fNPs synthesized from dopamine analogues were composed of carbon (C)-based organic nanoparticles.
다. XPS(X-ray photoelectron spectroscope) 스펙트럼 분석 all. X-ray photoelectron spectroscope (XPS) spectral analysis
도파민 및 이의 유사체 (a) LVD, (b) NPP, (c) HDA, (d)DA, (e) EPP 로부터 제조된 각각의 형광 나노입자를 구성하는 결합에너지를 분석하기 위하여 XPS 스펙트럼을 관찰하였다. 관찰 결과 도 3에 나타낸 바와 같이 각각의 도파민 유사체로부터 제조된 폴리도파민 기반의 형광 나노입자는 구조 내 탄소(C1s), 질소(N1s), 산소(O1s) 원소에 의한 케톤(C=O), C-C, C-N, C-NH2, C-OH의 결합 에너지를 모두 가지고 있었으며, 폴리도파민 구조의 카테콜(catechol), 도파퀴논(dopa quinone) 구조와 유사한 구조를 나타내고 있음을 확인하였다.XPS spectra were observed to analyze the binding energy of each fluorescent nanoparticle prepared from dopamine and its analogs (a) LVD, (b) NPP, (c) HDA, (d) DA, and (e) EPP. . As a result of observation, as shown in Figure 3, polydopamine-based fluorescent nanoparticles prepared from each dopamine analogue are ketones (C=O) and CC due to carbon (C1s), nitrogen (N1s), and oxygen (O1s) elements in the structure. , CN, C-NH 2 , and C-OH, and it was confirmed that it had a structure similar to the catechol and dopa quinone structures of the polydopamine structure.
3. 본 발명의 형광 나노입자(PFNPs)의 광학적 특성3. Optical properties of fluorescent nanoparticles (PFNPs) of the present invention
본 발명의 도파민 및 이의 유사체로부터 제조된 폴리도파민 기반 형광 나노입자는 염기성 수용액에서　　 고분자화(polymerization)가 진행되면서 에틸렌다이아민(EDA)와 같은 친핵체(nucleophile)로부터 화학적 감소(chemical degradation)에 의하여 형광이 발생하였다. 또한 중합(polymerization) 과정에서　　 단량체(monomer)의 구조적 차이에 따른 밴드갭 에너지의 혼성이 발생하며, 이는 단량체 의존적인 형광 파장의 다양성을 발생시킨다.Polydopamine-based fluorescent nanoparticles prepared from dopamine and its analogues of the present invention become fluorescent through chemical degradation from nucleophiles such as ethylenediamine (EDA) as polymerization progresses in basic aqueous solutions. This occurred. Additionally, during the polymerization process, bandgap energy hybridization occurs due to structural differences in monomers, which generates monomer-dependent fluorescence wavelength diversity.
가. 도파민 유사체의 구조적 차이에 따른 형광 나노입자의 형광 특성go. Fluorescence properties of fluorescent nanoparticles according to structural differences in dopamine analogues
　본 발명의 폴리도파민 기반 형광 나노입자는 제조되는 단량체의 구조적 차이에 의한 다양한 밴드갭(band gap) 혼성(hybridization)이 발생되므로, 따라서 다양한 도파민 유사체로부터 제조된 폴리도파민 기반 형광 나노입자는 최소 450nm부터 최대 650nm 이상의 파장에서 형광을 방출하는 것이 관찰되었다.(도 4) The polydopamine-based fluorescent nanoparticles of the present invention have various band gap hybridizations due to structural differences in the monomers from which they are produced. Therefore, polydopamine-based fluorescent nanoparticles prepared from various dopamine analogues have a range from at least 450 nm. Fluorescence was observed to be emitted at a wavelength of up to 650 nm or more (Figure 4).
나. PFNPs의 광학적 특징me. Optical characteristics of PFNPs
본 발명의 도파민 유사체로부터 제조된 폴리도파민 기반의 형광 나노입자는 밴드 갭(band gap)혼성으로 인한 다양한 band gap이 발생하며 이에 따른 서로 다른 형광 수명(fluorescence lifetime)을 나타내며, (a) Fluorescence lifetime 분석 결과, 각각의 도파민 유사체로부터 제조된 LVD, NPP, HDA, DA, EPP fNPs는 장파장대의 형광을 방출할수록 fluorescence lifetime이 증가하는 경향을 보였고, (b) 반응시간에 따른 형광 방출파장 분석 결과, 6-하이드록시도파민과(6-hydroxydopamine) 도파민(dopamine)으로부터 제조된 PFNPs를 제외하고 반응시간이 길어질수록 형광 방출파장의 적색편이(redshift)가 발생하였다.Polydopamine-based fluorescent nanoparticles prepared from dopamine analogues of the present invention generate various band gaps due to band gap hybridization and exhibit different fluorescence lifetimes accordingly. (a) Fluorescence lifetime analysis As a result, the LVD, NPP, HDA, DA, and EPP fNPs prepared from each dopamine analog showed a tendency for the fluorescence lifetime to increase as the fluorescence in the longer wavelength range was emitted. (b) Results of fluorescence emission wavelength analysis according to reaction time, 6- Except for PFNPs prepared from 6-hydroxydopamine and dopamine, a redshift in the fluorescence emission wavelength occurred as the reaction time increased.
다. UV-vis를 통한 광학적 분석 all. Optical analysis through UV-vis
본 발명의 도파민 유사체로부터 제조된 폴리도파민 기반의 형광 나노입자(PFNPs)의 흡광도(UV-vis spectroscopy) 분석 결과, C=C에 기인하는 π-π* 흡광 에너지가 280nm 내지 310nm에서 도파민 유사체로부터 제조된 모든 PFNPs에서 공통적으로 관찰되었다. 그러나, 구조적 차이에 따른 n-π* 흡광 에너지는 330nm 내지 450nm에서 폭 넓게 발견되었으며, 이는 PFNPs 합성 시 도파민 유사체 단량체의 분자구조 차이에 기인하는 것으로 판단된다.As a result of absorbance (UV-vis spectroscopy) analysis of polydopamine-based fluorescent nanoparticles (PFNPs) prepared from dopamine analogs of the present invention, π-π * absorption energy due to C=C was obtained from dopamine analogues at 280 nm to 310 nm. It was commonly observed in all PFNPs. However, n-π * absorption energy due to structural differences was found to be wide between 330 nm and 450 nm, which is believed to be due to differences in the molecular structure of dopamine analogue monomers during the synthesis of PFNPs.
4. 바이오이미징을 위한 본 발명의 형광 나노입자4. Fluorescent nanoparticles of the present invention for bioimaging
본 발명의 서로 다른 도파민 유사체로부터 제조된 폴리도파민 기반 형광 나노입자(PFNPs)는　낮은 독성 및 형광의 안정성 및 다양성으로 동물세포 및 동물에서 생체 이미징이 가능하며 이를 통한 바이오이미징 및 모니터링 기술을 제공할 수 있다. Polydopamine-based fluorescent nanoparticles (PFNPs) prepared from different dopamine analogues of the present invention enable bio-imaging in animal cells and animals due to low toxicity and stability and diversity of fluorescence, and can provide bio-imaging and monitoring technology through this. there is.
가. 본 발명의 형광 나노입자의 세포 내 형광 분석 go. Intracellular fluorescence analysis of fluorescent nanoparticles of the present invention
도파민 및 이의 유사체로부터 제조된 폴리도파민 기반 형광 나노입자를 MRC-5 세포에 각각 처리하고 24시간 후 형광 현미경의 DAPI, GFP, RFP 필터를 이용하여 세포의 형광 이미지를 관찰한 결과, LVD, NPP, HDA, DA, EPP fNPs 형광에 의한 파랑, 초록, 빨강의 형광의 다중 방출이 MRC-5 세포 내에서 관찰되었다.(도 6)MRC-5 cells were treated with polydopamine-based fluorescent nanoparticles prepared from dopamine and its analogs. 24 hours later, fluorescence images of the cells were observed using DAPI, GFP, and RFP filters of a fluorescence microscope. As a result, LVD, NPP, Multiple emission of blue, green, and red fluorescence due to HDA, DA, and EPP fNPs fluorescence was observed within MRC-5 cells (Figure 6).
나. 본 발명의 형광 나노입자의 생체 내 형광 이미징 me. In vivo fluorescence imaging of fluorescent nanoparticles of the present invention
도파민 및 이의 유사체로부터 제조된 폴리도파민 기반 형광 나노입자의 생체 내 형광 시그널을 확인하기 위하여, 생후 5주령이 지난 balb/c 누드(nude) 쥐(female)에 각각의 나노입자를 100uL(500ug/mL)의 용량으로 SC(subcutaneous) 주입 후 IVIS 생체 이미징 시스템 장비를 이용하여 Alexa514 및 Cy3 필터를 이용하여 생체 내 형광 이미징 분석을 진행하였다. To confirm the in vivo fluorescence signal of polydopamine-based fluorescent nanoparticles prepared from dopamine and its analogues, 100 uL (500 ug/mL) of each nanoparticle was administered to 5-week-old balb/c nude female mice. After subcutaneous injection at a dose of ), in vivo fluorescence imaging analysis was performed using Alexa514 and Cy3 filters using the IVIS bioimaging system equipment.
레보도파(LVD) 형광 나노입자는 푸른색, 노르네프피린(NPP) 형광 나노입자는 녹색, 6-하이드록시도파민(6-hydroxydopamine)은 노란색 내지 연두색, 도파민(dopamine)은 주황색, 그리고 에피네프린(epinephrine)은 붉은색의 형광 이미지를 각각 나타낼 수 있으나, 결과적으로, 도 7에서 확인할 수 있는 바와 같이 생체 내 주입된 나노입자 중 550nm 이상의 장파장 영역에서 형광 파장을 나타내는 HDA, DA, EPP fNPs가 생체 내에서 형광 시그널이 관찰되어 생체 내 형광 이미징에 효과적인 반면 형광 특성이 없는 PDA와 상대적으로 단파장의 형광을 방출하는 LVD, NPP, HDA의 형광 시그널은 관찰되지 않음을 확인하였다.Levodopa (LVD) fluorescent nanoparticles are blue, nornephpyrine (NPP) fluorescent nanoparticles are green, 6-hydroxydopamine is yellow to light green, dopamine is orange, and epinephrine. Each can display a red fluorescence image, but as a result, as can be seen in Figure 7, among the nanoparticles injected in vivo, HDA, DA, and EPP fNPs, which exhibit fluorescence wavelengths in the long wavelength region of 550 nm or more, fluoresce in vivo. It was confirmed that the signal was observed and was effective for in vivo fluorescence imaging, while the fluorescence signal of PDA, which has no fluorescence characteristics, and LVD, NPP, and HDA, which emit relatively short-wavelength fluorescence, was not observed.
다. 본 발명의 형광 나노입자의 세포독성 평가all. Cytotoxicity evaluation of fluorescent nanoparticles of the present invention
본 발명의 폴리도파민 형광 나노입자 기반의 PFNPs를 MRC-5 세포를 이용한 세포 독성 테스트를 진행하였다. 구체적으로, 5.0x103의 농도로 96-웰(well) 플레이트(plate)에 배양된 MRC-5 세포에 각각의 PFNPs를 50, 100, 200, 300, 400, 500ug/mL의 농도로 처리한 후 37℃ 습식 인큐베이터(CO2 5%)에서 12시간 배양하였다. 12시간 배양 후, PFNPs가 처리된 상층액을 제거하고 100uL 부피의 CCK-8 키트(kit) 용액을 30분 동안 처리하였다. 끝으로 처리가 완료된 96- 웰(well) 플레이트를 마이크로 리더(micro reader) 장비에서 450nm 흡광 파장을 측정하였다. PFNPs based on polydopamine fluorescent nanoparticles of the present invention were tested for cytotoxicity using MRC-5 cells. Specifically, MRC-5 cells cultured in a 96-well plate at a concentration of 5.0x10 3 were treated with each PFNPs at a concentration of 50, 100, 200, 300, 400, and 500 ug/mL. Cultured for 12 hours in a wet incubator (CO 2 5%) at 37°C. After 12 hours of incubation, the PFNPs-treated supernatant was removed and treated with a 100uL volume of CCK-8 kit solution for 30 minutes. Finally, the 450nm absorption wavelength of the processed 96-well plate was measured using a micro reader equipment.
결과적으로 도 8에서 확인할 수 있는 바와 같이 도파민 유사체로부터 제조된 폴리도파민 형광 나노입자 기반의 PFNPs는 100 내지 500ug/mL의 농도 구간에서 MRC-5에 대한 세포 생존율 85% 이상을 나타냄으로서 매우 낮은 독성으로 생체 적합성이 우수한 것으로 확인되었다.　　　 As a result, as can be seen in Figure 8, PFNPs based on polydopamine fluorescent nanoparticles prepared from dopamine analogues show a cell survival rate of more than 85% for MRC-5 in the concentration range of 100 to 500ug/mL, showing very low toxicity. It was confirmed to have excellent biocompatibility.
5. 본 발명의 폴리도파민 기반 형광 나노입자를 이용한 생체 접착 필름 제작 5. Production of bioadhesive film using polydopamine-based fluorescent nanoparticles of the present invention
자연계를 이용한 생체 모방 공학 중 생체 접착 분야는 바이오필름, 바이오패치, 스마트 패치 등 다양한 응용이 가능한 분야임. 그 대표적인 예로써 습식 접착의 경우는 홍합이 대표적인 연구 대상인데, 본 발명의 유사 홍합 접합 단백질인 폴리도파민 기반의 형광 나노입자는 표면에 생체 접착성이 있는 다수의 카테콜(catechol) 및 아민(amine)과 같은 작용기가 노출되어 있어 생체 접착성이 높으며, polydimethylsiloxane(PDMS)와 같은 생체 친화성 점탄성(viscoelastic) 고분자와　　 함께 이용함으로써 폴리도파민 기반의 생체 접착성이 우수한 있는 형광 필름(PFNPs-PDMS film)의 제조가 가능하였다.　　　 Among biomimetic engineering using the natural world, bioadhesion is a field that can be applied in a variety of ways, such as biofilms, biopatches, and smart patches. As a representative example, in the case of wet adhesion, mussels are a representative research target. The fluorescent nanoparticles based on polydopamine, a similar mussel adhesive protein of the present invention, contain a number of catechols and amines with bioadhesive properties on the surface. ), which has high bioadhesiveness due to exposed functional groups, and is used in combination with biocompatible viscoelastic polymers such as polydimethylsiloxane (PDMS) to create a polydopamine-based fluorescent film (PFNPs-PDMS film) with excellent bioadhesiveness. manufacturing was possible.
가. 폴리도파민 기반 형광나노입자를 이용한 생체 접착 필름 제작　 go. Production of bioadhesive film using polydopamine-based fluorescent nanoparticles
PDMS 필름의 제작을 위하여 사용된 실리콘 엘라스토머(silicone elastomer, SYLGARD 184)의 두 용액 베이스(base) 및 경화제(curing agent)를 10:1의 부피 비율로 섞어준 후 1mL의 혼합 용액을 실리콘 몰드(1cm x 1cm)에 넣어 80℃에서 약 3시간 동안 경화하였다. 연속적으로, 경화된 PDMS 필름을 100mg의 도파민 및 이의 유사체 (LVD, NPP, HDA, DA, EPP)가 녹아 있는 10mL의 Tris-HCl(pH 8.5) 완충 용액에 담그고 약 24시간 반응 후, 제작된 PFNPs-PDMS를 증류수로 3번 세척하여 건조하였다.　 도 9 (a)는 PFNPs-PDMS 필름의 제조 공적을 도식적으로 나타낸 것이고, 도 9 (b)는 제조된 PFNPs-PDMS 필름의 이미지를 나타낸 것이다.After mixing the two solutions of silicone elastomer (SYLGARD 184) used to produce the PDMS film, base and curing agent, at a volume ratio of 10:1, 1 mL of the mixed solution was placed in a silicone mold (1 cm). x 1cm) and cured at 80°C for about 3 hours. Continuously, the cured PDMS film was immersed in 10 mL of Tris-HCl (pH 8.5) buffer solution in which 100 mg of dopamine and its analogs (LVD, NPP, HDA, DA, EPP) were dissolved, and after reaction for about 24 hours, the produced PFNPs -PDMS was washed three times with distilled water and dried. Figure 9 (a) schematically shows the manufacturing process of the PFNPs-PDMS film, and Figure 9 (b) shows an image of the prepared PFNPs-PDMS film.
나. 폴리도파민 기반 형광 나노입자-생체 접착 필름의 생체 접착강도 실험을 위한 몰드 제작　 me. Mold fabrication for testing the bioadhesive strength of polydopamine-based fluorescent nanoparticle-bioadhesive film
본 발명의 PFNPs-PDMS 필름에 대하여 택 분리(Tack separation) 실험을 통해 생체 접착 능력을 평가하였다. 택 분리(Tack separation) 평가를 위하여 제작된 ABS 타입의 몰드는 3D 프린터를 통해 제작하였고, 이후 제작된 몰드에 PFNPs-PDMS 필름을 증착하기 위하여, 테이핑, PDMS 경화, PFNPs 도핑을 통하여 택 분리(Tack separation) 평가를 위한 시료를 제작하였다. 도 10은 이와 같은 PFNPs-PDMS 필름의 생체 접착강도 실험을 위한 몰드 제작 과정을 도식적으로 나타낸 것이다.The bioadhesion ability of the PFNPs-PDMS film of the present invention was evaluated through a tack separation experiment. An ABS type mold produced for tack separation evaluation was manufactured using a 3D printer, and in order to deposit a PFNPs-PDMS film on the manufactured mold, the tack was separated through taping, PDMS curing, and PFNPs doping. Separation) samples were produced for evaluation. Figure 10 schematically shows the mold manufacturing process for testing the bioadhesion strength of this PFNPs-PDMS film.
다. 폴리도파민 기반 형광 나노입자를 이용한 생체 접착 필름의 접착 강도 실험 all. Adhesion strength test of bioadhesive film using polydopamine-based fluorescent nanoparticles
PFNPs-PDMS 필름의 생체 접착성을 평가하기 위하여 생체물질로써 돼지 피부를 이용한 택 분리(Tack separation) 평가를 진행하였다. 실험 평가 방법으로, 돼지 피부가 접착되어 있는 ABS 몰드와 PFNPs-PDMS 필름이 증착되어 있는 ABS 몰드를 서로 마주보게 하면서 상온 조건에서 부착시킨 후 도 11과 같은 공정에 의해 UTM(universal testing machine)을 이용하여 평가를 진행하였다.To evaluate the bioadhesion of the PFNPs-PDMS film, a tack separation evaluation was conducted using pig skin as a biomaterial. As an experimental evaluation method, the ABS mold with the pig skin attached and the ABS mold with the PFNPs-PDMS film deposited on it are attached at room temperature while facing each other, and then a UTM (universal testing machine) is used through the process shown in FIG. 11. The evaluation was carried out.
라. 폴리도파민 기반 형광 나노입자를 이용한 생체 접착 필름의 접착 강도 la. Adhesion strength of bioadhesive film using polydopamine-based fluorescent nanoparticles
본 발명의 PFNPs-PDMS 필름의 UTM을 이용한 생체 접착 강도는 도 12 (a)와 같이 측정되어 계산하였다.The bioadhesion strength using UTM of the PFNPs-PDMS film of the present invention was measured and calculated as shown in Figure 12 (a).
결론적으로, 도 12 (b) 및 (c)와 같이 본 발명의 도파민 또는 이의 유사체 LVD, HDA, NPP, DA, EPP 나노입자를 이용하여 제조된 PFNPs-PDMS 필름은 기존에 보고되었던 폴리도파민-PDMS 필름과 비교하여 2 내지 3배 우수한 생체 접착성을 나타내는 것으로 확인되었다. In conclusion, as shown in Figures 12 (b) and (c), the PFNPs-PDMS film manufactured using dopamine or its analogue LVD, HDA, NPP, DA, and EPP nanoparticles of the present invention is similar to the previously reported polydopamine-PDMS. It was confirmed that it exhibits 2 to 3 times better bioadhesion compared to film.
마. 폴리도파민 기반 형광 나노입자를 이용한 생체 접착 필름의 형광을 통한 생체 내 모니터링 mind. In vivo monitoring through fluorescence of bioadhesive films using polydopamine-based fluorescent nanoparticles
본 발명의 PFNPs-PDMS 필름은 생체 접착성이 우수할 뿐만 아니라 생체 내 형광을 통한 모니터링이 가능한 것으로 확인되었다. 따라서 PFNPs-PDMS 필름을 쥐의 체내에 이식하여 생체 내 형광 모니터링을 진행하였다. 구체적으로, LVD, HDA, NPP, DA, EPP 나노입자로부터 제조된 PFNPs-PDMS 필름(0.5x0.5cm)을 생후 5주령이 지난 balb/c 누드(nude) 쥐의 표피(epidermis)에 이식한 뒤 24시간 후 생체영상장비(IVIS)를 이용하여 Alexa514 및 Cy3 필터에서 형광 이미지를 관찰하였다. It was confirmed that the PFNPs-PDMS film of the present invention not only has excellent bioadhesiveness but also enables monitoring through in vivo fluorescence. Therefore, PFNPs-PDMS film was implanted into the rat body and in vivo fluorescence monitoring was performed. Specifically, PFNPs-PDMS films (0.5x0.5cm) prepared from LVD, HDA, NPP, DA, and EPP nanoparticles were implanted into the epidermis of 5-week-old balb/c nude mice. After 24 hours, fluorescence images were observed through Alexa514 and Cy3 filters using an in vivo imaging system (IVIS).
결과적으로 도 13과 같이 폴리도파민(PDA)-PDMS 필름 및 LVD-PDMS 필름을 제외한 나머지 시료에서 생체 내 형광 이미지를 이용한 모니터링이 가능함을 확인하였다. 보다 상세하게, 본 발명의 PFNPs-PDMS 필름을 생체내(in vivo) 생체 이미징에 적용한 결과 폴리도파민(PDA)-PDMS 및 LVD-PDMS 필름은 단파장을 방출한 것으로 확인된 반면 EPP는 가장 장파장을　방출하는 것으로 확인하였으며, HDA, NPP, 및 EPP는 각각 구분이 필요한 생체 영역의 이미징에 사용될 수 있는 가능성을 확인하였다.As a result, as shown in Figure 13, it was confirmed that monitoring using in vivo fluorescence images was possible for the remaining samples except for the polydopamine (PDA)-PDMS film and the LVD-PDMS film. More specifically, as a result of applying the PFNPs-PDMS film of the present invention to in vivo bioimaging, it was confirmed that polydopamine (PDA)-PDMS and LVD-PDMS films emit short wavelengths, while EPP emits the longest wavelength. It was confirmed that HDA, NPP, and EPP each have the potential to be used for imaging of biological areas that require distinction.
특히 도 21에서 볼 수 있듯이, DA-PDMS 필름 및 EPP-PDMS 필름을 balb/c 누드(nude) 마우스 표피에 생체 표피 조직에 이식하여 형광 이미징을 15일 동안 관측한 결과, 이식 부위의 변형 및 형광의 감소 없이 제조된 각 각의 필름이 생체 영역에서 장기간 형광 이미징이 가능함을 확인하였다. In particular, as can be seen in Figure 21, the DA-PDMS film and EPP-PDMS film were implanted into the epidermis of a balb/c nude mouse and fluorescence imaging was observed for 15 days, resulting in deformation and fluorescence of the implantation site. It was confirmed that each film produced without a decrease in was capable of long-term fluorescence imaging in the biological area.
바. 폴리도파민 기반 형광 나노입자를 이용한 생체 접착 필름의 조직학적 독성 분석 bar. Histological toxicity analysis of bioadhesive films using polydopamine-based fluorescent nanoparticles
본 발명의 PFNPs-PDMS 필름을 체내 이식한 balb/c 누드(nude) 마우스의 표피를 절개하여 PFNPs-PDMS 필름의 조직학적 독성 분석을 진행하였다. 구체적으로, PFNPs-PDMS 필름을 이식한 마우스의 표피를 포름알데하이드(formaldehyde)에 고정한 후 H&E(hematoxylin and eosin staining)을 통해 표피 조직에 대한 독성 여부를 평가한 결과, 도 14에서 확인할 수 있는 바와 같이 모든 표피 조직에서 핵(nucleus) 및 세포질(cytosol)의 비정상화는 관찰되지 않았으며 이는 조직학적 독성이 매우 낮음을 의미한다.The epidermis of balb/c nude mice implanted with the PFNPs-PDMS film of the present invention was incised and histological toxicity analysis of the PFNPs-PDMS film was performed. Specifically, the epidermis of mice implanted with the PFNPs-PDMS film was fixed in formaldehyde and then assessed for toxicity to the epidermal tissue through H&E (hematoxylin and eosin staining), as can be seen in Figure 14. Abnormalities of the nucleus and cytosol were not observed in all epidermal tissues, indicating very low histological toxicity.
6. 본 발명의 폴리도파민 기반 형광나노입자의 암 세포 광열 치료 효과 6. Cancer cell photothermal treatment effect of polydopamine-based fluorescent nanoparticles of the present invention
가. 폴리도파민 기반의 형광 나노입자-PVA injectable 하이드로겔(hydrogel) 제작go. Production of polydopamine-based fluorescent nanoparticle-PVA injectable hydrogel
광열치료는 암 발생 부위에 나노입자와 같은 소재를 국부적으로 전달한 후 레이저를 조사하여 일정 온도의 열을 발생시킴으로써 암세포를 사멸시키는 치료법으로 기존 암 치료법인 수술이나 화학적 요법에서 발생하는 고통과 부작용을 줄일 수 있는 새로운 비침습적 암 치료법이다. 본 발명의 형광 나노입자는 poly(vinylalcohol)(PVA)과 같은 고분자와 결합하여 근적외선에 의한 광열 변환 효과를 나타내며 이로부터 암세포 사멸에 의한 암 치료가 가능한 주입형(injectable) 하이드로겔(hydrogel)을 제작하였다. 구체적으로, 25mL의 증류수에 녹아있는 0.25g의 PVA와 10mL의 증류수에 녹아있는 PFNPs(2.5mg/mL)를 혼합하여 95℃에서 3시간 가열하였다. 약 3시간의 겔화(gelation) 후, 혼합된 PFNPs-PVA 하이드로겔을 상온에서 냉각시켜 제조한 하이드로겔을 빛을 차단하여 보관하였다. Photothermal therapy is a treatment that kills cancer cells by locally delivering materials such as nanoparticles to the area where cancer occurs and then irradiating a laser to generate heat at a certain temperature. It reduces the pain and side effects caused by existing cancer treatments such as surgery or chemical therapy. It is a new non-invasive cancer treatment that can be used. The fluorescent nanoparticles of the present invention exhibit a photothermal conversion effect by near-infrared rays when combined with a polymer such as poly(vinylalcohol) (PVA), thereby producing an injectable hydrogel capable of treating cancer by killing cancer cells. did. Specifically, 0.25 g of PVA dissolved in 25 mL of distilled water and PFNPs (2.5 mg/mL) dissolved in 10 mL of distilled water were mixed and heated at 95°C for 3 hours. After gelation for about 3 hours, the hydrogel prepared by cooling the mixed PFNPs-PVA hydrogel at room temperature was stored by blocking light.
도 15는 본 발멸의 형광 나노입자를 이용한 하이드로겔 제작 및 광열 효과 메커니즘을 도시한 것이다. Figure 15 shows the hydrogel production and photothermal effect mechanism using the fluorescent nanoparticles of the present invention.
나. 폴리도파민 형광 나노입자-PVA 하이드로겔의 생체 외 광열 변환 효과me. In vitro photothermal conversion effect of polydopamine fluorescent nanoparticle-PVA hydrogel.
PVA 및 PFNPs로부터 각각 제조된 PFNPs-PVA 하이드로겔을 1.5mL의 투명 초자 용기에 넣어준 후,　1W 세기의 근적외선(808 nm) 레이저를 10분간 조사하여 근적외선 카메라를 통해 온도 변화를 관찰하였다.PFNPs-PVA hydrogels prepared from PVA and PFNPs were placed in a 1.5 mL transparent glass container, then irradiated with a 1W near-infrared (808 nm) laser for 10 minutes, and temperature changes were observed using a near-infrared camera.
결과적으로, PVA-hydrogel은 근적외선을 10분간 조사하여도 온도 변화가 관찰되지 않지만 PFNPs-PVA 하이드로겔에서는 최대 58.2℃까지 온도가 상승하는 것이 관찰되었다. 도 16은 (a) PVA-하이드로겔 및 도파민 유사체 (b) LVD, (c) NPP, (d) HDA, (e) DA, (f) EPP로부터 제조된 각 PFNPs-PVA 하이드로겔의 시간에 따른 광열 변환 효과를 나타낸 것이다. As a result, no temperature change was observed in PVA-hydrogel even when irradiated with near-infrared rays for 10 minutes, but in PFNPs-PVA hydrogel, the temperature was observed to rise up to 58.2°C. Figure 16 shows the time of each PFNPs-PVA hydrogel prepared from (a) PVA-hydrogel and dopamine analog (b) LVD, (c) NPP, (d) HDA, (e) DA, and (f) EPP. This shows the light-to-heat conversion effect.
이러한 결과에 의해 폴리도파민 기반의 다양한 유사체들의 서로 다른 구조적 특성으로, 근적외선 부근 흡광도 및 광열 변환 효과의 차이에 기인하는 것을 알 수 있다. These results show that the different structural properties of various polydopamine-based analogues are due to differences in absorbance near the near-infrared and photothermal conversion effects.
다. 폴리도파민 형광 나노입자-PVA 하이드로겔의 생체 내 광열 변환 효과all. In vivo photothermal conversion effect of polydopamine fluorescent nanoparticle-PVA hydrogel.
본 발명의 PFNPs-PVA 하이드로겔 100uL를 생후 6주 된 balb/c 누드(nude) 쥐(female)에 i.t (intratumoral) 주입 후, 1W 세기의 근적외선(808 nm) 레이저를 5분간 조사한 후 온도변화를　측정하였다. After i.t (intratumoral) injection of 100uL of the PFNPs-PVA hydrogel of the present invention into a 6-week-old balb/c nude female rat, the temperature change was measured after irradiating a 1W intensity near-infrared (808 nm) laser for 5 minutes. Measured.
도 17은 도파민 유사체 LVD, NPP, HDA, DA, EPP로부터 제조된 PFNPs-PVA 하이드로겔의　생체 내 광열 변환 효과를 나타낸 것으로, 결과적으로 PBS(phosphate buffer saline)가 주입된 쥐와는 다르게 LVD, NPP, HDA, DA, EPP-PVA 하이드로겔이 주입된 쥐에서만 최대 52.8℃까지 온도가 상승하였으며, 이는PFNPs-PVA 하이드로겔의 생체 내 광열 변환 효과에 의한 암 치료가 가능함을 의미한다.Figure 17 shows the in vivo photothermal conversion effect of PFNPs-PVA hydrogel prepared from dopamine analogues LVD, NPP, HDA, DA, and EPP. As a result, unlike rats injected with PBS (phosphate buffer saline), LVD, NPP , the temperature increased up to 52.8°C only in mice injected with HDA, DA, and EPP-PVA hydrogel, which means that cancer treatment is possible by the in vivo photothermal conversion effect of PFNPs-PVA hydrogel.
라. 폴리도파민 형광 나노입자-PVA의 암 치료 효과la. Cancer treatment effect of polydopamine fluorescent nanoparticles-PVA
PFNPs-PVA 하이드로겔 복합소재를 100uL의 부피로 4T1 암세포가 80mm3의 크기로 자란 6주령 balb/c 누드(nude) 쥐에 i.t(intratumoral) 주입 후 14일 동안 이틀 간격으로 근적외선을 조사하여 광열 치료를 통한 암세포의 활성 억제효과를 확인하였다.Photothermal treatment was performed by irradiating near-infrared rays every two days for 14 days after intratumoral injection of PFNPs-PVA hydrogel composite material into 6-week-old balb/c nude mice in which 4T1 cancer cells were grown to a size of 80 mm 3 in a volume of 100 uL. The effect of suppressing the activity of cancer cells was confirmed.
도 18은 (a) 근적외선 레이져 조사에 따른 시간 별 PFNPs-PVA 하이드로겔을 주입한 쥐의 온도변화, 14일 동안의 광열 치료에 따른 쥐의 (b) 체중 변화 및 (c) 암세포 부피의 변화를 나타낸 것으로, 결과적으로 PBS가 주입된 쥐와 비교하여 PFNPs-하이드로겔(LVD-PVA, NPP-PVA, HDA-PVA, DA-PVA, EPP-PVA)에 근적외선이 조사된 쥐는 5분 안에 온도가 높게 상승하였으며, 또한 14일간의 광열 치료 중 쥐의 체중 변화는 관찰되지 않으며 암 세포의 부피는 최대 6배 이상 감소하였다. 한편, PFNPs-하이드로겔의 암 치료 효과는 도파민 유사체의 종류에 따라 조금씩 상이한 효과를 보이며 온도가 높을수록 치료 효과가 증가하는 경향을 보였다.Figure 18 shows (a) temperature change in mice injected with PFNPs-PVA hydrogel over time according to near-infrared laser irradiation, (b) body weight change, and (c) change in cancer cell volume of mice following photothermal treatment for 14 days. As shown, compared to mice injected with PBS, the temperature of mice irradiated with near-infrared PFNPs-hydrogels (LVD-PVA, NPP-PVA, HDA-PVA, DA-PVA, EPP-PVA) was higher within 5 minutes. In addition, no change in the rat's body weight was observed during 14 days of photothermal treatment, and the volume of cancer cells decreased by up to 6 times or more. Meanwhile, the cancer treatment effect of PFNPs-hydrogel showed slightly different effects depending on the type of dopamine analogue, and the treatment effect tended to increase as the temperature increased.
마. 폴리도파민 형광 나노입자-PVA 하이드로겔의 광열 치료 후 암 세포의 조직학적 검사mind. Histological examination of cancer cells after photothermal treatment of polydopamine fluorescent nanoparticle-PVA hydrogel.
PFNPs-PVA 하이드로겔을 이용한 광열 치료 효과를 관찰하기 위하여, 광열 치료 14일 후 balb/c 누드(nude) 쥐의 암 세포를 절개하여 암 세포를 포름알데하이드(formaldehyde)에 고정한 후 H&E(hematoxylin and eosin) 염색을 분석하였다.To observe the effect of photothermal treatment using PFNPs-PVA hydrogel, cancer cells from balb/c nude mice were excised 14 days after photothermal treatment, the cancer cells were fixed in formaldehyde, and then treated with H&E (hematoxylin and eosin). ) Staining was analyzed.
도 19는 PFNPs-PVA 하이드로겔을 이용한 광열 치료 후 암 세포의 H&E 염색 결과를 나타낸 것으로, 결과적으로 PBS가 주입된 쥐(control)의 암 세포 조직에서 핵(nucleus) 및 세포질(cytosol)의 비정상화는 보이지 않은 반면 PFNPs-PVA 하이드로겔을 주입 후 광열 치료가 진행된 암 세포 조직에서는 광열 치료에 의한 핵 및 세포질의　수축 등 세포 사멸의 흔적이 발견되었다. Figure 19 shows the results of H&E staining of cancer cells after photothermal treatment using PFNPs-PVA hydrogel. As a result, abnormality of the nucleus and cytosol in the cancer cell tissue of mice (control) injected with PBS was observed. On the other hand, traces of cell death, such as shrinkage of the nucleus and cytoplasm due to photothermal treatment, were found in cancer cell tissue that underwent photothermal treatment after injection of PFNPs-PVA hydrogel.
바. 폴리도파민 형광 나노입자-PVA의 광열 치료 후 주요 장기 세포의 조직학적 검사bar. Histological examination of major organ cells after photothermal treatment of polydopamine fluorescent nanoparticles-PVA.
PFNPs-PVA 하이드로겔을 이용한 광열 치료 효과를 확인하기 위하여, 광열 치료 14일 후 balb/c 누드(nude) 쥐의 주요 장기인 심장, 폐, 간, 지라, 신장 조직세포를 절개하여 포름알데하이드(formaldehyde)에 고정한 후 H&E(hematoxylin and eosin) 염색을 분석하였다.To confirm the effect of photothermal treatment using PFNPs-PVA hydrogel, 14 days after photothermal treatment, tissue cells of the major organs of balb/c nude mice (heart, lung, liver, spleen, and kidney) were excised and treated with formaldehyde. ) and then analyzed by H&E (hematoxylin and eosin) staining.
도 20은 PFNPs-PVA 하이드로겔을 이용한 광열 치료 후 심장, 폐, 간, 지라, 신장 세포의 H&E 염색 결과를 나타낸 것으로, 결과적으로 PBS가 주입된 쥐(control)의 주요 장기조직과 마찬가지로 PFNPs-PVA 하이드로겔을 주입 후 광열 치료가 진행된 쥐의 주요 장기조직에서 핵(nucleus) 및 세포질(cytosol)의 비정상화는 보이지 않았으며, 이에 결론적으로 PFNPs-PVA 하이드로겔을 이용한 광열 치료는 심장, 폐, 간, 지라, 신장 조직에 독성을 미치지 않으며 암 세포 특이적인 억제 효과를 보이는 것을 확인할 수 있었다. Figure 20 shows the H&E staining results of heart, lung, liver, spleen, and kidney cells after photothermal treatment using PFNPs-PVA hydrogel. As a result, PFNPs-PVA, like the major organ tissues of mice (control) injected with PBS, Abnormalities of the nucleus and cytosol were not observed in the major organ tissues of mice that underwent photothermal treatment after injection of the hydrogel. In conclusion, photothermal treatment using PFNPs-PVA hydrogel was effective in the heart, lungs, and liver. It was confirmed that it was not toxic to spleen and kidney tissues and showed a specific inhibitory effect on cancer cells.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. This will be self-evident to those with ordinary knowledge in the field.

Claims (13)

  1. 도파민(dopamine) 또는 도파민 유사체를 염기성 용매 하에서 C1-C5알킬렌디아민과 혼합하는 단계를 포함하는, 형광 나노입자의 제조방법. A method for producing fluorescent nanoparticles, comprising mixing dopamine or a dopamine analog with C 1 -C 5 alkylenediamine in a basic solvent.
  2. 제1항에 있어서, 상기 C1-C5알킬렌디아민은 에틸렌디아민(ethylenediamine)인, 형광 나노입자의 제조방법.The method of claim 1, wherein the C 1 -C 5 alkylenediamine is ethylenediamine.
  3. 제1항에 있어서, 도파민 유사체는 레보도파민(levodopamine), 노르에피네프린(norepinephrine), 에피네프린(epinephrine), 및 6-하이드록시도파민(6-hydroxydopamine)으로 이루어진 그룹으로부터 선택되는 적어도 하나인, 형광 나노입자의 제조방법. The fluorescent nanoparticle of claim 1, wherein the dopamine analog is at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine. Manufacturing method.
  4. 제1항에 있어서, 상기 염기성 용매는 암모니아 수용액인, 형광 나노입자의 제조방법.The method of claim 1, wherein the basic solvent is an ammonia aqueous solution.
  5. 도파민(dopamine) 또는 도파민 유사체의 중합체인, 형광 나노입자. Fluorescent nanoparticles, which are polymers of dopamine or dopamine analogues.
  6. 제5항에 있어서, 도파민 유사체는 레보도파민(levodopamine), 노르에피네프린(norepinephrine), 에피네프린(epinephrine), 및 6-하이드록시도파민(6-hydroxydopamine)으로 이루어진 그룹으로부터 선택되는 적어도 하나인, 형광 나노입자.The fluorescent nanoparticle of claim 5, wherein the dopamine analog is at least one selected from the group consisting of levodopamine, norepinephrine, epinephrine, and 6-hydroxydopamine. .
  7. 제5항에 있어서, 상기 형광 나노입자는 50 내지 300nm의 입경인, 형광 나노입자. The fluorescent nanoparticle of claim 5, wherein the fluorescent nanoparticle has a particle size of 50 to 300 nm.
  8. 제5항에 있어서, 상기 형광 나노입자는 레보도파민 폴리머, 6-하이드록시도파민 폴리머 및 에피네프린 폴리머 입자 중 적어도 하나를 포함하는 형광 나노입자의 조합인, 형광 나노입자.The fluorescent nanoparticle of claim 5, wherein the fluorescent nanoparticle is a combination of fluorescent nanoparticles containing at least one of levodopamine polymer, 6-hydroxydopamine polymer, and epinephrine polymer particles.
  9. 제8항의 형광 나노입자를 포함하여 복수 영역의 이미징이 가능한, 이미징 조성물.An imaging composition capable of imaging multiple areas, including the fluorescent nanoparticle of claim 8.
  10. 제9항에 있어서, 상기 이미징 조성물은 상기 형광 나노입자를 포함하는 PDMS 필름의 형태인, 이미징 조성물.The imaging composition of claim 9, wherein the imaging composition is in the form of a PDMS film containing the fluorescent nanoparticles.
  11. 제5항 내지 제8항 중 어느 한 항의 형광 나노입자, PVA 및 물을 포함하는, 하이드로겔. A hydrogel comprising the fluorescent nanoparticles of any one of claims 5 to 8, PVA, and water.
  12. 제11항에 있어서, 상기 하이드로겔은 주입용(injectable) 하이드로겔인, 하이드로겔. The hydrogel of claim 11, wherein the hydrogel is an injectable hydrogel.
  13. 제11항에 있어서, 상기 하이드로겔은 광열치료용인, 하이드로겔.The hydrogel according to claim 11, wherein the hydrogel is for photothermal treatment.
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