Keloid prevention or treatment method using liquid phase plasma

Abstract

The present invention relates to a method of preventing or treating keloids using a liquid plasma. A liquid plasma according to the present invention is remarkably effective in inhibiting the generation and proliferation of keloids, thus being expected to be greatly utilized for the prevention and treatment of keloids.

Claims

1. A method of preparing a liquid plasma for preventing or treating keloids, the method comprising: (a) a step of charging a plasma generation apparatus with a carrier gas, wherein the carrier gas is prepared by mixing nitrogen and argon at a ratio of 15:2% by volume; (b) a step of supplying a voltage of 1 kV to 20 kV and a frequency of 10 to 30 kHz to the plasma generation apparatus to generate plasma; and (c) a step of irradiating a liquid material with the generated plasma, wherein the irradiation is carried out for 10 to 60 seconds per ml at a distance of 0.1 cm to 15 cm from a surface of a liquid material, wherein the liquid material of step (c) is water, saline, buffer, or medium.

2. A pharmaceutical composition, comprising a liquid plasma prepared according to the method according to claim 1.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is an oral formulation, a parenteral formulation, or a topical formulation.

4. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is used alone or in combination surgery, radiation therapy, hormonal therapy, chemotherapy, and methods of using biological response modifiers.

5. A method of preventing or treating keloids, the method comprising a step of administering the pharmaceutical composition according to claim 2 to a subject.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram illustrating a method of preparing a liquid plasma according to an embodiment of the present invention.

(2) FIG. 2 is a result illustrating NO generation effects in keloid fibroblasts treated with a liquid plasma or directly treated with plasma using various carrier gases according to an embodiment of the present invention.

(3) FIG. 3 is a result illustrating cell migration inhibition effect on keloid fibroblasts treated with a liquid plasma using various carrier gases according to an embodiment of the present invention.

(4) FIG. 4 is a result illustrating cell migration inhibition effects on keloid fibroblasts treated with liquid plasmas prepared using various plasma treatment times according to an embodiment of the present invention.

BEST MODE

(5) Hereinafter, the present invention will be described in more detail with reference to the following Examples. It will be apparent to those skilled in the art that Examples are merely for concretely explaining the invention and therefore, there is no intent to limit the invention to Examples.

EXAMPLE 1

Liquid Plasma (NTS) Preparation

(6) A liquid plasma was prepared using an atmospheric-pressure plasma generation apparatus including a quartz or ceramic tube, as a dielectric, and a non-thermal plasma source with multiple nozzles. The apparatus was provided with a gas supply nozzle with a diameter of less than 3 mm and was designed to generate uniform plasma of 1 inch size. A carrier gas was supplied at a flow rate of 10 L/min to the apparatus, and a bottom surface of a culture plate (12-well plate, TPP, Renner, Dannstadt, Germany) containing 2 ml of a cell medium was irradiated with plasma for 30 seconds per ml at a distance of 4 cm from the bottom surface. Here, a power supply of the plasma apparatus had specifications such as preferably a voltage of 1 to 20 kV and an average frequency 10 to 30 kHz, most preferably a voltage of 3 kV and an operation frequency of 25 kHz, but the present invention is not limited thereto. A schematic diagram of a method of preparing the liquid plasma is illustrated in FIG. 1.

EXAMPLE 2

Culture of Keloid Fibroblasts

(7) Fibroblasts isolated from skin tissue, diagnosed as keloid, spread beyond an original wound boundary for one or more years even after a wounded skin tissue was recovered were termed “keloid fibroblasts (KF).” KF was cultured in RPMI-1640 medium containing 10% by volume of FBS and 1% by volume of an antibiotic/antimicrobial under 5% by volume of CO.sub.2 in a 37° C. wet environment. Passage culture was performed when KF proliferated to a density of 80 to 90%. The passage was performed using trypsin. In examples of the present invention, cells of F2 to F7 generations were only used.

EXAMPLE 3

Confirmation of Nitric Oxide (NO) Generation Effect Upon Treatment of Keloid Fibroblasts with Plasma

(8) Primary keloid fibroblasts derived from humans were dispensed at a concentration of 1×10.sup.5 cells/ml in a 96-well plate and stabilized for 24 hours, followed by treatment with plasma. Particularly, cells were directly treated with plasma (direct plasma) for 30 seconds using the plasma apparatus described in Example 1, or treated with a liquid plasma prepared according to the method of Example 1. The methods were classified and summarized in Table 1 according to the type of used gases.

(9) TABLE-US-00001 TABLE 1 Direct plasma Non-thermal plasma treatment treated solution (NTS) Control No treatment Treatment with medium non-irradiated with plasma (general medium) N.sub.2 Use of nitrogen as carrier gas upon plasma generation Ar Use of argon as carrier gas upon plasma generation N.sub.2/Ar Use of mixture of nitrogen and argon, mixed in ratio of 1500:200, as carrier gas upon plasma generation

(10) After treating with plasma or a liquid plasma, additional culturing was performed for 24 hours. The culture medium was collected and centrifuged (12,000 rpm, 3 min). Nitric oxide (NO) was quantified using the Griess reagent method. NO, generated from arginine (L-arginine) by a nitric oxide synthase, is known to play a role in promoting cellular metabolism through an increase in intracellular cGMP. In particular, 100 μl of a medium of each group and 100 μl of a Griess solution (5% (v/v) phosphoric acid containing 1% sulfanilamide and 0.1% naphthylethylenediamine) were dispensed in a 96-well plate and allowed to react for 10 minutes, followed by measuring an absorbance at 550 nm. Results are shown in FIG. 2.

(11) From the experimental results, it was confirmed that {circle around (1)} a NO generation amount in the case treated with the liquid plasma was not reduced compared to the case in which cells were directly treated with plasma, {circle around (2)} both the case treated with the liquid plasma and the case directly treated with plasma exhibited NO generation increase effects compared to a control, and {circle around (3)} NO generation increase effect in the case using the mixed nitrogen/argon gas as a carrier gas was more significant than the cases using nitrogen or argon alone.

EXAMPLE 4

Confirmation of Cell Migration Inhibition Effect Upon Treatment of Keloid Fibroblasts with Plasma

(12) To investigate the cell migration inhibition effect of keloid fibroblasts treated with a liquid plasma, the keloid fibroblasts were cultured at a density of 5×10.sup.5/well in a 12-well culture plate. The center of cells forming a monolayer sheet was scraped with a tip, followed by washing to remove cell debris due to the scraping. Treatment with a liquid plasma was performed as summarized in Table 1. After performing additional culture for 24 hours, cellular migration was measured. Results are shown in FIG. 3.

(13) As an experimental result, it was confirmed that all groups treated with the liquid plasma exhibited significant inhibition of cell migration, particularly the case using the mixed nitrogen/argon gas as a carrier gas exhibited greatly significant cell migration effect, compared to the control treated with a general medium.

EXAMPLE 5

Confirmation of Plasma Treatment Time-Dependent Cell Migration Inhibition Effect in Keloid Fibroblasts

(14) Based on the experimental results of Example 4, cell migration inhibition effects of liquid plasmas prepared using the mixed nitrogen/argon gas as a carrier gas, but varying a plasma treatment time were investigated. Experiments were investigated in the same manner as in Example 4.

(15) As experimental results, it was confirmed that the cell migration inhibition effect on keloid fibroblasts was slight in the case of the liquid plasma prepared by irradiating a medium with plasma for 10 seconds, whereas the cell migration inhibition effect on keloid fibroblasts in the case of the liquid plasma prepared by irradiating a medium with plasma for 30 seconds was significant, compared to the control (general medium). However, cell proliferation was similarly maintained in all groups, which indicates that the cell migration inhibition is not due to a decrease in cell proliferation. Results are shown in FIG. 4.

(16) From the results of Examples 1 to 5, it was confirmed that, when plasma was applied in the form of a liquid plasma, the therapeutic effect on keloids was not reduced, compared to the case wherein cells were directly treated with plasma. However, considering that, when treated with a liquid plasma, cell damage is reduced, there is no risk of skin damage such as burns due to a wrong apparatus operation by a user, and plasma may be uniformly applied even to a wide and curved area, compared to the case of directly treating a wounded skin lesion with plasma, a liquid plasma is more medically useful than direct plasma treatment.

(17) In addition, it was confirmed that therapeutic effects differed depending upon the type of carrier gas used to generate plasma, and, when plasma was generated using mixed nitrogen/argon gas as a carrier gas, treatment effects on keloids were superior to the cases of using a nitrogen or argon gas alone.