WO2018056636A1 - Complex for treating inflammation and wounds which is capable of dual drug delivery - Google Patents

Complex for treating inflammation and wounds which is capable of dual drug delivery Download PDF

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Publication number
WO2018056636A1
WO2018056636A1 PCT/KR2017/010071 KR2017010071W WO2018056636A1 WO 2018056636 A1 WO2018056636 A1 WO 2018056636A1 KR 2017010071 W KR2017010071 W KR 2017010071W WO 2018056636 A1 WO2018056636 A1 WO 2018056636A1
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complex
present
inflammation
silica nanoparticles
wound
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PCT/KR2017/010071
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French (fr)
Korean (ko)
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박철용
홍진기
유재찬
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동국대학교 산학협력단
중앙대학교 산학협력단
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Priority claimed from KR1020170115438A external-priority patent/KR101980002B1/en
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Publication of WO2018056636A1 publication Critical patent/WO2018056636A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles

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  • the present invention relates to an inflammation and wound treatment complex, and more specifically, two or more therapeutic substances such as nitric oxide and hyaluronic acid included in the treatment complex are simultaneously applied to a wound site (especially, ocular surface damage, etc.), A therapeutic complex capable of wound healing and inflammation control.
  • wound dressing is used to effectively treat wounds caused by physical damage such as wounds and traumas.
  • Wound dressings require the ability to maintain adequate moisture at the contact surface with wounds, the ability to control wound secretions, ease of attachment and removal of dressings to wounds, and air and water vapor penetration between external wounds. Sex, insulation of the wound area against the outside, resistance to the invasion of bacteria, non-toxic to the human body, excellent mechanical properties are required.
  • the present invention has been made to solve the above problems, the inventors of the present invention invented the inflammatory and wound treatment complex excellent in wound healing effect, by trapping the drug in a double, the drug is released in close contact with the wound site.
  • It provides a complex for treating inflammation and wound, comprising hyaluronic acid bound to the silica nanoparticles.
  • Another object of the present invention is to provide a third object of the present invention.
  • It provides a complex for treating inflammation and wound, comprising hyaluronic acid bound to the silica nanoparticles.
  • the therapeutic substance may be nitric oxide.
  • the silica nanoparticles may have a size of 30 to 400nm.
  • the silica nanoparticles may not exhibit cell damage.
  • the therapeutic material, silica nanoparticles and hyaluronic acid can increase cell viability.
  • a contact lens including a complex for treating inflammation and wound, comprising hyaluronic acid bound to silica nanoparticles.
  • the therapeutic material through the silica nanoparticles and hyaluronic acid, two or more therapeutic materials are applied to the wound site (especially, ocular surface damage, etc.) at the same time, control the release rate This is possible, which may allow for effective wound and inflammation control.
  • the therapeutic complex is believed to be able to heal trauma by application of eye, skin, oral mucosa, nasal mucosa, internal organs, and the like, in particular, made in the form of contact lenses effectively when intractable inflammation or trauma of the eye surface occurs It is expected to be applicable.
  • Figure 1 shows a state of treating the inflammation of the eye surface using the inflammation and wound treatment complex according to an embodiment of the present invention.
  • Figure 2 shows the results confirming the effect of increasing cell survival according to the concentration of nitric oxide used as a therapeutic material in the inflammation and wound treatment complex according to an embodiment of the present invention.
  • nitric oxide used as a therapeutic substance promotes wound healing in mechanical and chemical corneal trauma of experimental animals (Balb / c mice). The result is a reduction in corneal haze.
  • Figure 6 shows the result of confirming the effect of increasing corneal cell survival according to hyaluronic acid in the inflammation and wound treatment complex according to an embodiment of the present invention.
  • 9 and 10 show the results of confirming the change in the p-mTOR signal according to the treatment of the silica nanoparticles in the inflammation and wound treatment complex according to an embodiment of the present invention.
  • 11 to 14 show the results of the electron microscope photograph of the cell state when the silica nanoparticles treated with the corneal stromal cells in the inflammation and wound treatment complex according to an embodiment of the present invention.
  • 15 and 16 show the results of confirming the change of vimentin after 24 hours treatment of 50, 100 and 150 nm-sized silica nanoparticles to corneal cells at a concentration of 100 ⁇ g / mL.
  • Figure 17 shows the result of reduced IL-6, an inflammatory cytokine of corneal epithelial cells by silica nanoparticles in the inflammation and wound treatment complex of the present invention.
  • Figure 18 shows the result of the reduction of the inflammatory cytokine IL-1 ⁇ of corneal epithelial cells by silica nanoparticles in the inflammation and wound treatment complex of the present invention.
  • FIG. 19 shows the form of a composite film synthesized with the nitric oxide-releasing nanoparticles and hyaluronic acid of the present invention.
  • Figure 21 shows the epithelial regeneration effect of the composite membrane synthesized nitric oxide releasing nanoparticles and hyaluronic acid of the present invention in a rabbit model damaged corneal epithelial cells.
  • FIG. 22 shows a decrease in epithelial cell defect diameter by a composite membrane synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid in a rabbit model in which corneal epithelial cells are damaged.
  • Inflammation and wound treatment complex by containing a therapeutic material in each of the silica nanoparticles and hyaluronic acid, it may be in contact with the site where the trauma occurred, to deliver the therapeutic material to the trauma site.
  • a therapeutic material in each of the silica nanoparticles and hyaluronic acid, it may be in contact with the site where the trauma occurred, to deliver the therapeutic material to the trauma site.
  • the therapeutic material is applied to the affected area to induce tissue regeneration, such as to treat wounds and to control inflammation.
  • tissue regeneration such as to treat wounds and to control inflammation.
  • nitrogen oxide it is preferable to use nitrogen oxide as the therapeutic material, but the desired therapeutic effect If it is possible to increase the therapeutic effect by using a therapeutic substance corresponding to the, it can be used without limitation.
  • the nitric oxide affects the removal of free radicals (Oxygen frree radical) of the wound, inflammation control, angiogenesis and epithelialization, so that the wound can be treated quickly.
  • the inflammation and wound treatment complex according to an embodiment of the present invention was packaged to the nitrogen oxide using silica nanoparticles in order to clinically apply nitrogen oxide of the gas component as a therapeutic material.
  • the nanoparticles are released by the nitrogen oxide contained in the recording conditions under the reaction with water, thereby, it may be possible to control the discharge rate of the nitric oxide.
  • the silica nanoparticles may have a size of 30 to 400 nm, but is not limited thereto.
  • hyaluronic acid promotes epithelial regeneration and helps to heal wounds, and is a component that normally exists in vivo, the eye surface is used as the main material of artificial tears.
  • the present invention two or more therapeutic substances are applied simultaneously to the wound site (especially, ocular surface damage, etc.), thereby making it possible to more effectively induce wound healing and inflammation control.
  • the silica nanoparticles and hyaluronic acid containing the therapeutic material are preferably combined to form a complex.
  • the bonding between the silica nanoparticles and hyaluronic acid may be in the form of fusion, mixing, coating or lamination, and at this time, a form based on hyaluronic acid and including the nanoparticles may be preferable. have. Therefore, when the complex is in direct contact with the wound site, when hyaluronic acid is released from the wound site and the treatment is started, the embedded silica nanoparticles are exposed to the wound site, thereby releasing a therapeutic material therein. Healing may proceed concurrently with the Lonic acid, which may facilitate the healing of wounds.
  • the inflammation and wound treatment complex according to an embodiment of the present invention in tight contact with the wound site, for this purpose, the complex is preferably made of a semi-solid. As a result, it may deform into close contact with the wound over time.
  • the contact lens may be prepared by a conventional manufacturing method in the art, and at this time, it is preferable to be prepared including the inflammation and wound treatment complex of the present invention.
  • the contact lens may be made of a material and / or a shape of a contact lens generally used, such as a hard lens, a soft lens, or a toric lens, but is not limited thereto.
  • the contact lens of the present invention may include various bases and / or additives necessary and suitable for its manufacture, and may further include known inflammation and / or wound healing materials within the scope of not impairing the effect thereof.
  • FIG. 1 illustrates a state using an inflammation and wound treatment complex according to an embodiment of the present invention.
  • the inflammation and wound treatment complex according to an embodiment of the present invention in order to use the inflammation and wound treatment complex according to an embodiment of the present invention to cover the inflammation and wound treatment complex in the position where the trauma occurred.
  • the traumatic site may be skin, eyeball, oral mucosa, nasal mucosa, internal organs, and the like, preferably eyeball.
  • the treatment begins at the trauma site by hyaluronic acid of the inflammation and wound treatment complex, and when the hyaluronic acid starts to be released, the embedded silica nanoparticles are exposed to the trauma site, and when dissolved by moisture,
  • the therapeutic material of nitric oxide is discharged and the wound healing process can be progressed simultaneously with hyaluronic acid. As the wound healing is promoted, the therapeutic material can be continuously discharged, and the treatment of the persistent wound can be possible.
  • an experiment was conducted to confirm the effect of increasing the survival of corneal cells according to the nitric oxide used as the therapeutic material, corneal cells of hyaluronic acid The effect of increasing the survival of was confirmed, and also the effect of increasing the survival of corneal cells and reducing the inflammatory cytokine according to the concentration and size of the silica nanoparticles (see Examples 1 to 4).
  • the corneal cell treatment effect of the composite membrane synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid was confirmed (see Example 5).
  • nitric oxide used as a therapeutic substance in order to confirm the wound healing effect of nitric oxide used as a therapeutic substance, the effect of increasing the survival of corneal epithelium, epilepsy and endothelial cells according to the concentration of nitric oxide was confirmed. More specifically, NaNO2 liberating nitric oxide was added to the cellular corneal cells at a concentration up to 1000 nM, and after 24 hours, cell viability was confirmed.
  • mice In order to further confirm the wound healing effect of nitric oxide, experiments were conducted using an animal model (Balb / c mice). More specifically, after the chemical burn was induced with 0.1 N NaOH in 10 Balb / c mice, the nitric oxide treatment group received 10 ⁇ M of nitric oxide four times a day, and the percentage of corneal epithelial defect and The degree of turbidity was tracked from day 1 to day 5 and compared with the control group.
  • the corneal epithelial damage rate was added to the corneas of five Balb / c mice using a surgical knife, and then, 100% of nitric oxide was applied to the nitric oxide treatment group four times a day to restore epithelial defects due to the recovery of epithelial damage.
  • 100% of nitric oxide was applied to the nitric oxide treatment group four times a day to restore epithelial defects due to the recovery of epithelial damage.
  • 5 days in the experiment was compared to the control.
  • the hyaluronic acid was added to the corneal endothelial cell culture by concentration (0, 0.1, 1, 10, 100 and 1000 ⁇ g / ml), and the corneal stromal cell culture Hyaluronic acid was added by concentrations (0, 10, 50 100, 500 and 1000 ng / ml) to confirm cell viability after 24 and 48 hours.
  • the nanoparticles of 50, 100 and 150 nm size were treated in concentration-specific (0, 25, 50 and 100 ⁇ g / ml) cell cultures to give CCK- 8 assay and experiments to confirm the membrane destruction indicator LDH was carried out.
  • the silica nanoparticles were treated to the corneal stromal cells. At this time, it was treated by silica nanoparticle size (50, 100, 150 nm) at a concentration of 100 ⁇ g / ml, and observed the cell state with an electron microscope.
  • the corneal stromal cells were differentiated into myofibroblasts after treatment for 24 hours at a concentration of 100 ⁇ g / ml by silica nanoparticles (50, 100, 150 nm) for 24 hours. The change of vimentin was confirmed.
  • mice (Balb / c) caused bacterial keratitis in the cornea by Pseudomonas aeruginosa were treated with nitric oxide-releasing silica nanoparticles for 7 days to treat inflammatory cytokines.
  • An experiment was conducted to confirm the cain.
  • a composite lens in the form of a contact lens synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid was prepared as shown in FIG. 19.
  • the nitric oxide releasing composite membrane prepared as described above stably released nitric oxide for up to 30 hours.
  • the composite membrane according to the present invention significantly increased epithelial regeneration after mechanical corneal epithelial injury.

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Abstract

The present invention relates to a therapeutic complex for treating inflammation and wounds, more specifically, relates to a therapeutic complex for treating inflammation and wounds in which two or more therapeutic substances are simultaneously applied to an injured part, particularly a damaged ocular surface part, through therapeutic substance-loaded silica nanoparticles and hyaluronic acid, thereby promoting wound healing. Accordingly, the therapeutic complex for treating inflammation and wounds according to the present invention is expected to be able to be usefully used in treating an external injury to the eyes, skin, oral mucosa, nasal mucous membrane, internal organs or the like or infectious diseases thereof.

Description

이중 약물전달이 가능한 염증 및 상처 치료 복합체Inflammatory and wound healing complex with dual drug delivery
본 발명은 염증 및 상처 치료 복합체에 관한 것으로서, 보다 구체적으로는, 상기 치료 복합체에 포함된 산화질소 및 히알루론산 등 두 가지 이상의 치료 물질이 상처 부위 (특히, 안구 표면 손상 등)에 동시에 적용됨으로써, 상처 치료 및 염증 조절이 가능한 치료 복합체에 관한 것이다.The present invention relates to an inflammation and wound treatment complex, and more specifically, two or more therapeutic substances such as nitric oxide and hyaluronic acid included in the treatment complex are simultaneously applied to a wound site (especially, ocular surface damage, etc.), A therapeutic complex capable of wound healing and inflammation control.
일반적으로 창상, 외상 등과 같은 물리적 손상에 따른 상처를 효과적으로 치료하기 위해서는 드레싱 (Wound dressing)을 사용한다. 상처 치료용 드레싱이 가져야 할 특성으로는 상처와의 접촉면에서 적당한 습기의 유지능력이 필요하고, 상처 분비물의 조절능력, 상처에 대한 드레싱의 부착 및 제거의 용이성, 외부의 상처부위간의 공기 및 수증기 관통성, 외부에 대한 상처부위의 단열성, 박테리아의 침입에 대한 저항성, 인체에 대한 무독성, 우수한 기계적 물성 등이 요구된다.Generally, wound dressing is used to effectively treat wounds caused by physical damage such as wounds and traumas. Wound dressings require the ability to maintain adequate moisture at the contact surface with wounds, the ability to control wound secretions, ease of attachment and removal of dressings to wounds, and air and water vapor penetration between external wounds. Sex, insulation of the wound area against the outside, resistance to the invasion of bacteria, non-toxic to the human body, excellent mechanical properties are required.
한편, 각막의 손상이나, 수술 시 안구 표면질환의 치료를 위해서는 10-0 나일론 봉합을 시행하는 등 고난이도의 외과 수술 기술이 필요하고, 수술 시간이 오래 걸리는 등의 단점이 있었다. 또한, 봉합에 따른 여러 부작용, 예컨대 봉합 농양, 육아종 형성, 조직 괴사 등이 발생하는 실정이다.On the other hand, in order to treat corneal damage or ocular surface disease during surgery, a high degree of surgical technique such as 10-0 nylon suture is required, and the operation time is long. In addition, a number of side effects such as suture abscess, granulomatous formation, tissue necrosis, etc. occur due to suture.
따라서, 안구를 포함하는 상처 부위에 밀착 접촉하여 효과적으로 약물을 전달하여 상처 치유의 촉진이 가능한 장치의 개발이 주요한 과제의 대상이 되고 있고, 이에 대한 연구가 이루어지고 있으나(한국등록특허 10-1132625), 아직은 미비한 실정이다.Therefore, the development of a device capable of promoting the healing of wounds by effectively delivering a drug in close contact with the wound site including the eye has been the subject of a major problem, and research on this has been made (Korea Patent No. 10-1132625) It is still inadequate.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 이중으로 약물을 포집하고, 상처 부위에 밀착 접촉하여 약물이 방출됨으로써, 상처 치유 효과가 뛰어난 염증 및 상처 치료 복합체를 발명하였다.The present invention has been made to solve the above problems, the inventors of the present invention invented the inflammatory and wound treatment complex excellent in wound healing effect, by trapping the drug in a double, the drug is released in close contact with the wound site.
이에, 본 발명의 목적은Thus, the object of the present invention
치료 물질이 담지되는 실리카 나노입자; 및Silica nanoparticles carrying a therapeutic material; And
상기 실리카 나노입자와 결합하는 히알루론산을 포함하는, 염증 및 상처 치료용 복합체를 제공하는 것이다.It provides a complex for treating inflammation and wound, comprising hyaluronic acid bound to the silica nanoparticles.
본 발명의 다른 목적은Another object of the present invention
실리카 나노입자와 결합하는 히알루론산을 포함하는, 염증 및 상처 치료용 복합체를 포함하는 콘택트렌즈를 제공하는 것이다.It is to provide a contact lens comprising a complex for treating inflammation and wound, comprising hyaluronic acid bound to silica nanoparticles.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위하여, 본 발명은In order to achieve the above object, the present invention
치료 물질이 담지되는 실리카 나노입자; 및Silica nanoparticles carrying a therapeutic material; And
상기 실리카 나노입자와 결합하는 히알루론산을 포함하는, 염증 및 상처 치료용 복합체를 제공한다.It provides a complex for treating inflammation and wound, comprising hyaluronic acid bound to the silica nanoparticles.
바람직하게는, 상기 치료 물질은, 산화질소일 수 있다.Preferably, the therapeutic substance may be nitric oxide.
바람직하게는, 상기 실리카 나노입자는, 30 내지 400nm의 크기를 가질 수 있다.Preferably, the silica nanoparticles may have a size of 30 to 400nm.
바람직하게는, 상기 실리카 나노입자는, 세포 손상을 나타내지 않을 수 있다.Preferably, the silica nanoparticles may not exhibit cell damage.
바람직하게는, 상기 치료 물질, 실리카 나노입자 및 히알루론산은, 세포 생존율을 증가시킬 수 있다.Preferably, the therapeutic material, silica nanoparticles and hyaluronic acid can increase cell viability.
상기 목적을 달성하기 위하여, 본 발명은In order to achieve the above object, the present invention
실리카 나노입자와 결합하는 히알루론산을 포함하는, 염증 및 상처 치료용 복합체를 포함하는 콘택트렌즈를 제공한다.Provided is a contact lens including a complex for treating inflammation and wound, comprising hyaluronic acid bound to silica nanoparticles.
본 발명에 따른 염증 및 상처 치료 복합체는 치료 물질이 실리카 나노입자 및 히알루론산을 통해서, 두 가지 이상의 치료 물질이 상처 부위 (특히, 안구 표면 손상 등)에 동시에 적용되며, 치료 물질의 방출 속도의 조절이 가능한 바, 이로 인해 효과적으로 상처 및 염증 조절이 가능할 수 있다.Inflammation and wound treatment complex according to the present invention, the therapeutic material through the silica nanoparticles and hyaluronic acid, two or more therapeutic materials are applied to the wound site (especially, ocular surface damage, etc.) at the same time, control the release rate This is possible, which may allow for effective wound and inflammation control.
또한, 상기 치료 복합체는 안구, 피부, 구강점막, 비강 점막, 내부 장기 등의 적용하여 외상 치유가 가능할 것으로 판단되며, 특히, 콘택트렌즈 형상으로 제작되어 안구 표면의 난치성 염증 또는 외상이 발생할 때, 효과적으로 적용이 가능할 것으로 기대된다.In addition, the therapeutic complex is believed to be able to heal trauma by application of eye, skin, oral mucosa, nasal mucosa, internal organs, and the like, in particular, made in the form of contact lenses effectively when intractable inflammation or trauma of the eye surface occurs It is expected to be applicable.
도 1은 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체를 이용하여 안구 표면의 염증을 치료하는 상태를 나타낸 것이다.Figure 1 shows a state of treating the inflammation of the eye surface using the inflammation and wound treatment complex according to an embodiment of the present invention.
도 2는 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서, 치료 물질로 사용되는 산화질소의 농도에 따른 세포 생존 증가 효과를 확인한 결과를 나타낸 것이다.Figure 2 shows the results confirming the effect of increasing cell survival according to the concentration of nitric oxide used as a therapeutic material in the inflammation and wound treatment complex according to an embodiment of the present invention.
도 3 내지 5는 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서, 치료 물질로 사용되는 산화질소가 실험동물 (Balb/c 생쥐)의 기계적 각막 외상과 화학적 각막 외상에서 상처 치유를 촉진하고 각막 혼탁을 감소시키는 결과를 나타낸 것이다.3 to 5 shows that in the inflammation and wound treatment complex according to an embodiment of the present invention, nitric oxide used as a therapeutic substance promotes wound healing in mechanical and chemical corneal trauma of experimental animals (Balb / c mice). The result is a reduction in corneal haze.
도 6은 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서, 히알루론산에 따른 각막 세포 생존 증가 효과를 확인한 결과를 나타낸 것이다.Figure 6 shows the result of confirming the effect of increasing corneal cell survival according to hyaluronic acid in the inflammation and wound treatment complex according to an embodiment of the present invention.
도 7 및 8은 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서 실리카 나노입자에 의한 각막 상피세포의 생존률이 실리카 나노입자에 의하여 감소하지 않으며 세포막 파괴 정도를 나타내는 LDH도 증가하지 않는 결과를 나타낸 것이다.7 and 8 show that the survival rate of corneal epithelial cells by silica nanoparticles in the inflammation and wound treatment complex according to an embodiment of the present invention is not reduced by silica nanoparticles, and LDH, which indicates the degree of cell membrane destruction, does not increase. It is shown.
도 9 및 10은 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서 실리카 나노입자의 처리에 따른 p-mTOR 신호의 변화를 확인한 결과를 나타낸 것이다.9 and 10 show the results of confirming the change in the p-mTOR signal according to the treatment of the silica nanoparticles in the inflammation and wound treatment complex according to an embodiment of the present invention.
도 11 내지 14는 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체에서 실리카 나노입자를 각막 간질세포에 처리하였을 때, 세포 상태를 전자 현미경으로 촬영한 결과를 나타낸 것이다.11 to 14 show the results of the electron microscope photograph of the cell state when the silica nanoparticles treated with the corneal stromal cells in the inflammation and wound treatment complex according to an embodiment of the present invention.
도 15 및 16은 100 μg/mL 농도의 각막세포에 50, 100 및 150 nm크기의 실리카 나노입자를 24시간 처리한 후, vimentin의 변화를 확인한 결과를 나타낸 것이다.15 and 16 show the results of confirming the change of vimentin after 24 hours treatment of 50, 100 and 150 nm-sized silica nanoparticles to corneal cells at a concentration of 100 μg / mL.
도 17은 본 발명의 염증 및 상처 치료 복합체에서 실리카 나노입자에 의한 각막 상피세포의 염증성 사이토카인인 IL-6가 감소된 결과를 나타낸 것이다.Figure 17 shows the result of reduced IL-6, an inflammatory cytokine of corneal epithelial cells by silica nanoparticles in the inflammation and wound treatment complex of the present invention.
도 18은 본 발명의 염증 및 상처 치료 복합체에서 실리카 나노입자에 의한 각막 상피세포의 염증성 사이토카인인 IL-1β가 감소된 결과를 나타낸 것이다.Figure 18 shows the result of the reduction of the inflammatory cytokine IL-1β of corneal epithelial cells by silica nanoparticles in the inflammation and wound treatment complex of the present invention.
도 19는 본 발명의 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막의 형태를 나타낸 것이다.19 shows the form of a composite film synthesized with the nitric oxide-releasing nanoparticles and hyaluronic acid of the present invention.
도 20은 본 발명의 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막에서 30시간 까지 안정적으로 산화질소가 방출된다는 결과를 나타낸 것이다.20 shows the results that nitrogen oxide is stably released up to 30 hours in the composite film synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid of the present invention.
도 21은 각막 상피세포가 손상된 토끼 모델에서, 본 발명의 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막의 상피재생 효과를 나타낸 것이다. Figure 21 shows the epithelial regeneration effect of the composite membrane synthesized nitric oxide releasing nanoparticles and hyaluronic acid of the present invention in a rabbit model damaged corneal epithelial cells.
도 22는 각막 상피세포가 손상된 토끼 모델에서, 본 발명의 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막에 의한 상피세포 결손 직경의 감소를 나타낸 것이다. FIG. 22 shows a decrease in epithelial cell defect diameter by a composite membrane synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid in a rabbit model in which corneal epithelial cells are damaged.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일실시예에 따른 염증 및 상처 치료 복합체는, 실리카 나노입자 및 히알루론산에 각각에 치료 물질을 내포함으로써, 외상이 발생한 부위에 접촉시켜 상기 치료 물질을 외상 부위로 전달할 수 있다. 이와 같은 구성을 채택함으로써, 치료 기간 동안 외상 부위에 접촉한 상태로 효율적인 약물의 전달이 가능하며, 이로 인해, 빠른 상처 치유가 가능할 수 있을 것으로 기대된다.Inflammation and wound treatment complex according to an embodiment of the present invention, by containing a therapeutic material in each of the silica nanoparticles and hyaluronic acid, it may be in contact with the site where the trauma occurred, to deliver the therapeutic material to the trauma site. By adopting such a configuration, it is expected that efficient drug delivery is possible in contact with the traumatic site during the treatment period, thereby enabling rapid wound healing.
이하에서는, 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체를 구성하는 각각의 구성요소에 대하여 상세히 설명하기로 한다.Hereinafter, each component constituting the inflammation and wound treatment complex according to an embodiment of the present invention will be described in detail.
본 발명에 있어서, 치료 물질은 환부에 적용하여 조직의 재생을 유도하는 등 상처 치료 및 염증 조절을 하기 위한 물질로서, 이 때, 상기 치료 물질로는 산화질소를 사용하는 것이 바람직하나, 원하는 치료 효과에 대응하는 치료 물질을 사용하여 치료 효과를 높일 수 있다면, 이에 제한 없이 사용 가능하다. 이 때, 상기 산화질소는 상처 부위의 활성산소 (Oxygen frree radical) 제거, 염증 조절, 혈관형성 및 상피화에 영향을 미치는바, 상처를 신속하게 치료할 수 있다.In the present invention, the therapeutic material is applied to the affected area to induce tissue regeneration, such as to treat wounds and to control inflammation. At this time, it is preferable to use nitrogen oxide as the therapeutic material, but the desired therapeutic effect If it is possible to increase the therapeutic effect by using a therapeutic substance corresponding to the, it can be used without limitation. At this time, the nitric oxide affects the removal of free radicals (Oxygen frree radical) of the wound, inflammation control, angiogenesis and epithelialization, so that the wound can be treated quickly.
다만, 상기 산화질소는 기체 성분으로 이루어져 있기 때문에 임상적으로 적용함에 있어서 어려움이 있었다. 이에, 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체는 기체 성분의 산화질소를 치료 물질로써 임상에 적용하기 위해서, 실리카 나노 입자를 사용하여 상기 산화질소를 포장하였다. 이 때, 상기 나노 입자는 물과 반응하는 조건에서 녹음으로써 내포된 산화질소가 유리되는바, 이로 인해, 상기 산화질소의 배출 속도의 조절이 가능할 수 있다. 더욱이, 상기 실리카 나노입자는, 30 내지 400nm의 크기를 가질 수 있으나, 이에 제한되는 것은 아니다.However, since the nitric oxide is composed of a gas component, there was a difficulty in clinical application. Thus, the inflammation and wound treatment complex according to an embodiment of the present invention was packaged to the nitrogen oxide using silica nanoparticles in order to clinically apply nitrogen oxide of the gas component as a therapeutic material. At this time, the nanoparticles are released by the nitrogen oxide contained in the recording conditions under the reaction with water, thereby, it may be possible to control the discharge rate of the nitric oxide. Moreover, the silica nanoparticles may have a size of 30 to 400 nm, but is not limited thereto.
본 발명에 있어서, 히알루론산 (hyaluronic acid)은 상피재생을 촉진하여 상처 치유에 도움을 주며, 생체 내에 정상적으로 존재하는 성분으로서, 안구 표면의 경우 인공눈물의 주재료로 사용되고 있다.In the present invention, hyaluronic acid (hyaluronic acid) promotes epithelial regeneration and helps to heal wounds, and is a component that normally exists in vivo, the eye surface is used as the main material of artificial tears.
한편, 본 발명은 두 가지 이상의 치료 물질이 상처 부위 (특히, 안구 표면 손상 등)에 동시에 적용되어, 상처 치료 및 염증 조절을 보다 효과적으로 유도할 수 있게 한다. 이를 위해서 상기 치료 물질을 내포하는 실리카 나노 입자 및 히알루론산은 결합하여 복합체를 형성하는 것이 바람직하다.On the other hand, the present invention, two or more therapeutic substances are applied simultaneously to the wound site (especially, ocular surface damage, etc.), thereby making it possible to more effectively induce wound healing and inflammation control. To this end, the silica nanoparticles and hyaluronic acid containing the therapeutic material are preferably combined to form a complex.
이 때, 상기 실리카 나노 입자 및 히알루론산 간의 결합이란, 융합, 혼합, 코팅 또는 적층 등의 형태로 이루어질 수 있으며, 이 때, 히알루론산을 기반으로 하고, 상기 나노 입자가 포함되는 형태가 바람직할 수 있다. 이로 인해, 상기 복합체가 상처 부위와 직접적으로 접촉하면, 히알루론산이 상기 상처 부위에서 유리되어 치료가 시작되면 내포되어 있던 실리카 나노 입자가 상처 부위에 노출되어 내부에 있던 치료 물질이 유리되어, 상기 히알루론산과 동시에 치유가 진행될 수 있으며, 이로 인해 상처 치유의 촉진이 가능할 수 있다.In this case, the bonding between the silica nanoparticles and hyaluronic acid may be in the form of fusion, mixing, coating or lamination, and at this time, a form based on hyaluronic acid and including the nanoparticles may be preferable. have. Therefore, when the complex is in direct contact with the wound site, when hyaluronic acid is released from the wound site and the treatment is started, the embedded silica nanoparticles are exposed to the wound site, thereby releasing a therapeutic material therein. Healing may proceed concurrently with the Lonic acid, which may facilitate the healing of wounds.
한편, 상기 치유 과정이 진행됨에 있어서, 상처 부위에 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체가 견고하게 밀착 접촉되는 것이 바람직하며, 이를 위해서 상기 복합체는 반고형으로 이루지는 것이 바람직하다. 이로 인해 시간이 지나감에 따라 상처 부위에서 밀착 접촉된 형태로 변형될 수 있다. 특히, 판모양 또는 콘택트렌즈 형상으로 이루어져 안구에 적용할 경우 안구 표면의 난치성 염증 등의 치료에도 효과적으로 이용될 수 있다. 상기 콘택트렌즈는 당업계의 통상적인 제조방법으로 제조될 수 있으며, 이 때, 본 발명의 염증 및 상처 치료 복합체를 포함하여 제조되는 것이 바람직하다. 상기 콘택트렌즈는 하드렌즈, 소프트렌즈 또는 토릭렌즈 등 일반적으로 사용되는 콘택트렌즈의 재질 및/또는 형태로 제조될 수 있으나, 이에 한정되지 않는다.On the other hand, in the progress of the healing process, it is preferable that the inflammation and wound treatment complex according to an embodiment of the present invention in tight contact with the wound site, for this purpose, the complex is preferably made of a semi-solid. As a result, it may deform into close contact with the wound over time. In particular, when applied to the eye consisting of a plate-like or contact lens shape can be effectively used for the treatment of refractory inflammation of the eyeball. The contact lens may be prepared by a conventional manufacturing method in the art, and at this time, it is preferable to be prepared including the inflammation and wound treatment complex of the present invention. The contact lens may be made of a material and / or a shape of a contact lens generally used, such as a hard lens, a soft lens, or a toric lens, but is not limited thereto.
본 발명의 콘택트렌즈는 그 제조에 필요하고 적절한 각종 기제 및/또는 첨가물을 포함할 수 있으며, 그 효과를 떨어트리지 않는 범위 내에서 공지의 염증 및/또는 상처 치료 물질을 더 포함할 수 있다.The contact lens of the present invention may include various bases and / or additives necessary and suitable for its manufacture, and may further include known inflammation and / or wound healing materials within the scope of not impairing the effect thereof.
도 1은 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체를 사용하는 상태를 도시한 것이다. 도 1에 도시된 바와 같이, 본 발명의 일실시예에 따른 염증 및 상처 치료 복합체를 사용하기 위해서는 외상이 발생한 위치에 상기 염증 및 상처 치료 복합체를 덮어준다. 이 때, 외상 부위는 피부, 안구, 구강점막, 비강 점막, 내부 장기 등일 수 있으며, 바람직하게는 안구일 수 있다. 이 후, 상기 염증 및 상처 치료 복합체의 히알루론산에 의해서 외상 부위에서 치료가 시작이 되고, 히알루론산이 유리되기 시작하면, 내포되어 있던 실리카 나노입자가 외상 부위에 노출되고, 수분에 의해서 녹게 되면 내부의 치료 물질인 산화 질소가 배출되어 히알루론산과 동시에 상처 치유 과정이 진행될 수 있는바, 상처 치유가 촉진되면서, 치료 물질이 지속적으로 배출이 가능한바, 지속적인 상처 부위의 치료가 가능할 수 있다.1 illustrates a state using an inflammation and wound treatment complex according to an embodiment of the present invention. As shown in Figure 1, in order to use the inflammation and wound treatment complex according to an embodiment of the present invention to cover the inflammation and wound treatment complex in the position where the trauma occurred. At this time, the traumatic site may be skin, eyeball, oral mucosa, nasal mucosa, internal organs, and the like, preferably eyeball. Subsequently, the treatment begins at the trauma site by hyaluronic acid of the inflammation and wound treatment complex, and when the hyaluronic acid starts to be released, the embedded silica nanoparticles are exposed to the trauma site, and when dissolved by moisture, The therapeutic material of nitric oxide is discharged and the wound healing process can be progressed simultaneously with hyaluronic acid. As the wound healing is promoted, the therapeutic material can be continuously discharged, and the treatment of the persistent wound can be possible.
본 발명의 일실시예에서는, 치료 물질에 따른 상처 부위의 치유 효과를 확인하기 위해서, 치료 물질로서 사용되는 산화 질소에 따른 각막 세포의 생존 증가 효과를 확인하는 실험을 진행하였으며, 히알루론산의 각막 세포의 생존 증가 효과를 확인하였고, 또한, 실리카 나노 입자의 농도 및 크기에 따른 각막 세포의 생존 증가 효과 및 염증성 사이토카인 감소 효과를 확인하였다 (실시예 1 내지 4 참조).In one embodiment of the present invention, in order to confirm the healing effect of the wound site according to the therapeutic material, an experiment was conducted to confirm the effect of increasing the survival of corneal cells according to the nitric oxide used as the therapeutic material, corneal cells of hyaluronic acid The effect of increasing the survival of was confirmed, and also the effect of increasing the survival of corneal cells and reducing the inflammatory cytokine according to the concentration and size of the silica nanoparticles (see Examples 1 to 4).
또한, 본 발명의 일실시예에서는, 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막의 각막 세포 치료 효과를 확인하였다(실시예 5 참조).In addition, in one embodiment of the present invention, the corneal cell treatment effect of the composite membrane synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid was confirmed (see Example 5).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[[ 실시예Example ]]
실시예Example 1. 산화질소에 따른 상처 치료 효과 확인 1. Confirmation of wound healing effect by nitric oxide
1-1. in vitro 각막 세포에서의 산화질소 효과 확인1-1. Confirmation of nitric oxide effect in in vitro corneal cells
본 발명에 따른 염증 및 상처 치료 복합체에 있어서, 치료 물질로 사용되는 산화질소의 상처 치유 효과를 확인하기 위해서, 산화질소의 농도에 따른 각막 상피, 간질 및 내피세포의 생존 증가 효과를 확인하였다. 보다 구체적으로, 산화질소를 유리하는 NaNO2를 상기 세포 각막 세포에 1000nM 까지의 농도로 첨가하고 24 시간 후, 세포 생존율을 확인하였다.In the inflammation and wound treatment complex according to the present invention, in order to confirm the wound healing effect of nitric oxide used as a therapeutic substance, the effect of increasing the survival of corneal epithelium, epilepsy and endothelial cells according to the concentration of nitric oxide was confirmed. More specifically, NaNO2 liberating nitric oxide was added to the cellular corneal cells at a concentration up to 1000 nM, and after 24 hours, cell viability was confirmed.
그 결과, 도 2에 도시된 바와 같이, 세포 생존이 유의하게 증가하고 있는 것을 확인할 수 있었다.As a result, as shown in Figure 2, it was confirmed that the cell survival is significantly increased.
1-2. In 1-2. In vivovivo 동물모델에서의 산화질소 효과 확인 Identification of nitric oxide effects in animal models
산화질소의 상처 치유 효과를 더욱 확인하기 위해서, 동물 모델 (Balb/c 생쥐)을 이용한 실험을 진행하였다. 보다 구체적으로, Balb/c 생쥐 10마리의 각막에 0.1 N의 NaOH를 이용하여 화학적 화상을 유발한 후, 산화질소 치료군에는 산화질소 10μM을 하루 4회 점안하고, 각막 상피 결손부위의 % 및 각막의 혼탁 정도를 1일에서 5일까지 추적하여 대조군과 비교하는 실험을 진행하였다.In order to further confirm the wound healing effect of nitric oxide, experiments were conducted using an animal model (Balb / c mice). More specifically, after the chemical burn was induced with 0.1 N NaOH in 10 Balb / c mice, the nitric oxide treatment group received 10 μM of nitric oxide four times a day, and the percentage of corneal epithelial defect and The degree of turbidity was tracked from day 1 to day 5 and compared with the control group.
그 결과, 도 3에 도시된 바와 같이, 산화질소 치료군에서 각막결손부위의 %가 유의하게 낮은 것을 확인할 수 있었으며, 또한, 도 4에 도시된 바와 같이, 산화질소 치료군에서 각막 혼탁을 감소시키는 결과를 보였다.As a result, as shown in FIG. 3, it was confirmed that the percentage of corneal defects in the nitric oxide treatment group was significantly lower, and as shown in FIG. 4, the corneal haze was reduced in the nitric oxide treatment group. Seemed.
더욱이, Balb/c 생쥐 5마리의 각막에 수술칼을 이용하여 각막 상피 손상율을 100% 가한 후, 산화질소 치료군에는 산화질소 100μM을 하루 4회 점안하여 상피 손상 회복에 따른 상피 결손 %를 1일에서 5일까지 추적하여 대조군과 비교하는 실험을 진행하였다.Furthermore, the corneal epithelial damage rate was added to the corneas of five Balb / c mice using a surgical knife, and then, 100% of nitric oxide was applied to the nitric oxide treatment group four times a day to restore epithelial defects due to the recovery of epithelial damage. Followed up to 5 days in the experiment was compared to the control.
그 결과, 도 5에 도시된 바와 같이, 산화질소 치료군에서 유의하게 빠른 각막 상피 치유가 관찰되었다. As a result, significantly faster corneal epithelial healing was observed in the nitric oxide treatment group, as shown in FIG. 5.
이에, 실험동물 (Balb/c생쥐)의 기계적 각막 외상과 화학적 각막 외상에서 상처 치유 촉진 효과를 확인하였다.Thus, the effects of promoting wound healing on mechanical and chemical corneal trauma of experimental animals (Balb / c mice) were confirmed.
실시예Example 2. 히알루론산 ( 2. Hyaluronic Acid ( HyaluronicHyaluronic acid)의 각막 세포 생존 증가 효과 확인 acidic effect on corneal cell survival
본 발명에 따른 히알루론산의 치유 효과를 확인하기 위해서, 각막 내피세포 배양액에 상기 히알루론산을 농도별로(0, 0.1, 1, 10, 100 및 1000 ㎍/㎖) 첨가하였고, 각막 간질세포 배양액에 상기 히알루론산을 농도별로 (0, 10, 50 100, 500 및 1000 ng/㎖) 첨가하여 24시간 및 48시간 후 세포 생존율을 확인하였다.In order to confirm the healing effect of hyaluronic acid according to the present invention, the hyaluronic acid was added to the corneal endothelial cell culture by concentration (0, 0.1, 1, 10, 100 and 1000 ㎍ / ㎖), and the corneal stromal cell culture Hyaluronic acid was added by concentrations (0, 10, 50 100, 500 and 1000 ng / ml) to confirm cell viability after 24 and 48 hours.
그 결과, 도 6에 도시된 바와 같이, 히알루론산에 의해서 각막 내피 세포 및 간질 세포의 세포 생존이 유의하게 증가하는 것을 확인할 수 있었다.As a result, as shown in Figure 6, it was confirmed that the hyaluronic acid significantly increased cell survival of corneal endothelial cells and stromal cells.
실시예Example 3. 실리카 나노입자의 유해성 확인 3. Identification of hazards of silica nanoparticles
본 발명에 따른 실리카 나노 입자가 각막 세포의 미치는 영향을 확인하기 위해서, 50, 100 및 150 ㎚ 크기의 나노입자를 농도별 (0, 25, 50 및 100 ㎍/㎖) 세포 배양액에 처리하여 CCK-8 assay 및 세포막 파괴지표인 LDH를 확인하는 실험을 진행하였다.In order to determine the effect of the silica nanoparticles according to the present invention on the corneal cells, the nanoparticles of 50, 100 and 150 nm size were treated in concentration-specific (0, 25, 50 and 100 μg / ml) cell cultures to give CCK- 8 assay and experiments to confirm the membrane destruction indicator LDH was carried out.
그 결과, 도 7에 도시된 바와 같이, 24시간 후, 세포 생존력 (CCK-8 kit로 측정)이 유의하게 증가하여 각막 상피세포의 생존률이 실리카 나노입자에 의하여 감소하지 않았으며, 도 8에 도시된 바와 같이, LDH는 증가하지 않았는바, 실리카 나노입자에 의한 세포막 파괴가 나타나지 않는 것을 확인할 수 있었다.As a result, as shown in Figure 7, after 24 hours, the cell viability (measured by the CCK-8 kit) significantly increased so that the survival rate of corneal epithelial cells did not decrease by silica nanoparticles, as shown in Figure 8 As shown, LDH did not increase, and it was confirmed that cell membrane destruction by silica nanoparticles did not appear.
또한, 도 9 및 도 10에 도시된 바와 같이, 실리카 나노입자의 농도에 따라 각막 상피 세포에서 활성화된 p-mTOR가 유의하게 증가되는 것을 확인하였다.In addition, as shown in Figure 9 and 10, it was confirmed that the activated p-mTOR in the corneal epithelial cells significantly increased according to the concentration of the silica nanoparticles.
더욱이, 실리카 나노입자가 각막 세포의 미치는 영향을 더욱 확인하기 위해서, 도 11 내지 도 14에 도시된 바와 같이, 실리카 나노입자를 각막 간질세포에 처리하였다. 이때, 100 ㎍/㎖ 농도에 실리카 나노입자 크기별로 (50, 100, 150 ㎚) 처리하였으며, 세포 상태를 전자 현미경으로 관찰하였다.Furthermore, in order to further confirm the effect of the silica nanoparticles on the corneal cells, as shown in Figs. 11 to 14, the silica nanoparticles were treated to the corneal stromal cells. At this time, it was treated by silica nanoparticle size (50, 100, 150 nm) at a concentration of 100 ㎍ / ㎖, and observed the cell state with an electron microscope.
그 결과, 도 11 내지 도 14에 도시된 바와 같이, 각 크기별 나노입자를 세포의 노출하여도 세포의 구조가 잘 유지되며, 미토콘드리아와 핵막의 손상도 없는 것을 확인하였다.As a result, as shown in Figures 11 to 14, even when exposed to the nanoparticles of each size of the cell structure is well maintained, it was confirmed that no damage to the mitochondria and nuclear membrane.
이에 더하여, 나노 입자에 따른 각막 혼탁이 발생하는지 확인하기 위하여, 100 ㎍/㎖ 농도에 실리카 나노입자 크기별로 (50, 100, 150 ㎚) 24시간 처리하여 각막 간질세포의 근섬유세포로의 분화 표지자인 vimentin의 변화를 확인하였다.In addition, in order to determine whether corneal haze occurs according to the nanoparticles, the corneal stromal cells were differentiated into myofibroblasts after treatment for 24 hours at a concentration of 100 μg / ml by silica nanoparticles (50, 100, 150 nm) for 24 hours. The change of vimentin was confirmed.
그 결과, 도 15 및 도 16에 도시된 바와 같이, 상기 실리카 나노입자를 24시간 처리하여도 vimentin이 증가하지 않았는바, 각막 혼탁 예방 효과가 있음을 확인할 수 있었다.As a result, as shown in FIG. 15 and FIG. 16, even when the silica nanoparticles were treated for 24 hours, vimentin did not increase, and thus it was confirmed that the corneal haze prevention effect was obtained.
실시예 4. 산화질소 방출 실리카 나노입자의 유효성 확인Example 4 Validation of Nitric Oxide Release Silica Nanoparticles
본 발명에 따른 실리카 나노 입자가 각막 세포의 미치는 영향을 확인하기 위해서, 녹농균에 의해 각막에 세균성 각막염이 유발된 생쥐(Balb/c)를 산화질소 방출 실리카 나노입자를 이용하여 7일간 치료하여 염증성 사이토카인을 확인하는 실험을 진행하였다.In order to confirm the effect of the silica nanoparticles according to the present invention on the corneal cells, the mice (Balb / c) caused bacterial keratitis in the cornea by Pseudomonas aeruginosa were treated with nitric oxide-releasing silica nanoparticles for 7 days to treat inflammatory cytokines. An experiment was conducted to confirm the cain.
그 결과, 도 17 및 도 18에 도시된 바와 같이, 각막에서 발현되는 주요 염증성 사이토카인인 IL-6 및 IL-1β가 유의하게 감소함을 확인하였다.As a result, as shown in Figure 17 and 18, it was confirmed that the major inflammatory cytokines IL-6 and IL-1β expressed in the cornea significantly decreased.
실시예 5. 산화질소 방출 복합막의 유효성 확인Example 5 Validation of Nitric Oxide Release Composite Membranes
5-1. 복합막 제작5-1. Composite film production
본 발명에 따른 산화질소 방출 나노입자 및 히알루론산을 합성한 콘택트렌즈 형태의 복합막을 도 19과 같이 제작하였다. A composite lens in the form of a contact lens synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid was prepared as shown in FIG. 19.
상기와 같이 제작한 산화질소 방출 복합막은 도 20에 도시된 바와 같이, 30시간 까지 안정적으로 산화질소를 방출하였다.As shown in FIG. 20, the nitric oxide releasing composite membrane prepared as described above stably released nitric oxide for up to 30 hours.
5-2. 5-2. In vivoIn vivo 동물모델에서의 복합막 효과 확인 Checking the effect of composite membranes on animal models
본 발명에 따른 산화질소 방출 나노입자 및 히알루론산을 합성한 복합막의 효과를 확인하기 위하여, 토끼 각막에 수술칼을 이용하여 지름 8mm의 각막상피를 제거한 후, 실험군 (산화질소 히알루론산 복합막) 7마리 및 대조군 (무치료) 7마리의 각막 상피의 재생 속도를 비교하는 실험을 진행하였다. In order to confirm the effect of the composite membrane synthesized with nitric oxide-releasing nanoparticles and hyaluronic acid according to the present invention, after removing the corneal epithelium of 8mm diameter using a surgical knife on the rabbit cornea, experimental group (nitric oxide hyaluronic acid composite membrane) 7 Experiments were conducted to compare the regeneration rate of the corneal epithelium of the rats and the control (7 treatments).
그 결과, 외상 후 4일에 측정한 각막 재생속도는 대조군에 비하여 실험군에서 유의하게 빠름을 확인하였다(도 21).As a result, the corneal regeneration rate measured at 4 days after trauma was significantly faster in the experimental group compared to the control group (Fig. 21).
또한, 도 22에 도시된 바와 같이, 외상 후 4일에 측정한 잔존 각막 상피 결손의 크기 (직경)는 대조군 (control)에 비하여 복합막군 (study)에서 유의하게 작음을(p=0.020, Student's T test) 확인하였다.In addition, as shown in FIG. 22, the size (diameter) of residual corneal epithelial defects measured at 4 days after trauma was significantly smaller in the study group than the control group (p = 0.020, Student's T). test) confirmed.
즉, 본 발명에 따른 복합막은 기계적 각막 상피 손상 후 상피재생을 유의하게 증가시킨다는 것을 확인하였다.That is, it was confirmed that the composite membrane according to the present invention significantly increased epithelial regeneration after mechanical corneal epithelial injury.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (6)

  1. 치료 물질이 담지되는 실리카 나노입자; 및Silica nanoparticles carrying a therapeutic material; And
    상기 실리카 나노입자와 결합하는 히알루론산을 포함하는, 염증 및 상처 치료용 복합체.Comprising hyaluronic acid that binds to the silica nanoparticles, inflammation and wound treatment complex.
  2. 제1항에 있어서, 상기 치료 물질은, 산화질소인 것을 특징으로 하는, 염증 및 상처 치료용 복합체.The complex of claim 1, wherein the therapeutic substance is nitric oxide.
  3. 제1항에 있어서, 상기 실리카 나노입자는, 30 내지 400nm의 크기를 갖는 것을 특징으로 하는, 염증 및 상처 치료용 복합체.According to claim 1, The silica nanoparticles, characterized in that having a size of 30 to 400nm, inflammation and wound treatment complex.
  4. 제1항에 있어서, 상기 실리카 나노입자는, 세포 손상을 나타내지 않는 것을 특징으로 하는, 염증 및 상처 치료용 복합체.According to claim 1, The silica nanoparticles, characterized in that not showing cell damage, inflammation and wound treatment complex.
  5. 제1항에 있어서, 상기 치료 물질, 실리카 나노입자 및 히알루론산은, 세포 생존율을 증가시키는 것을 특징으로 하는, 염증 및 상처 치료용 복합체.The complex of claim 1, wherein the therapeutic substance, silica nanoparticles, and hyaluronic acid increase cell viability.
  6. 제 1항의 복합체를 포함하는 콘택트렌즈.A contact lens comprising the complex of claim 1.
PCT/KR2017/010071 2016-09-22 2017-09-14 Complex for treating inflammation and wounds which is capable of dual drug delivery WO2018056636A1 (en)

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KR1020170115438A KR101980002B1 (en) 2016-09-22 2017-09-08 Inflammation and wound treatment complex for controlled delivery of a dual-loaded drug
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CN104448407A (en) * 2014-12-12 2015-03-25 青岛中皓生物工程有限公司 Highly oxygen-permeable material based on marine biological substances as well as preparation and application of highly oxygen-permeable material

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