KR100817327B1 - Method of Surface Treatment with Improved Cell Adhesive Property by Pulsed Plasma Polymerization and a Carrier for Cell Adhesion Using the Same - Google Patents

Abstract

The present invention relates to a surface treatment method for maximizing cell adhesion using pulsed plasma. More specifically, the present invention relates to a surface treatment method and a cell adhesion carrier using the same, by controlling a duty cycle in a frequency range of 1000 Hz or less to have a polymer surface having excellent cell adhesion.

The present invention polymerizes a thin layer of several micrometers or less on a polymer film such as polystyrene or polyethylene terephthalate or a three-dimensional object to change only the surface properties of the bulk material without changing the properties of the bulk material, such as an amine such as allylamine. By using the material of the system as a precursor, it is possible to manufacture a carrier for cell adhesion excellent in cell adhesion and cell growth control can be widely used in the development of various medical devices or biomaterial products.

Pulsed Plasma Polymerization, Surface Treatment, Allylamine, Polymer Medical Devices, Plasma Deposition

Description

Method of Surface Treatment with Improved Cell Adhesive Property by Pulsed Plasma Polymerization and a Carrier for Cell Adhesion Using the Same}

The present invention relates to a surface treatment method of a polymer resin substrate that maximizes cell adhesion using pulsed plasma. More specifically, the present invention relates to a surface treatment method of a polymer resin substrate and a cell adhesion carrier using the same to control a duty cycle in a frequency range of 1000 Hz or less to have a polymer surface having excellent cell adhesion.

In order to take advantage of biological activities such as animal cells, plant cells, microorganisms, enzymes, etc., methods of adhering or immobilizing them have been developed. Such techniques ultimately reproduce artificial living tissues that can replace human tissues and organs, thereby transplanting them into the body. It can be applied to biological tissue engineering aimed at maintaining, improving or restoring the function of the human body, for example, musculoskeletal system (bone, cartilage, ligaments, teeth, etc.), cardiovascular system (heart valve, blood vessel, etc.), It can be applied to skin, nervous system (neural induction pipe, etc.), urogenital system (urethra, bladder, kidney, etc.) organs.

Generally, organic polymers in the form of gels, such as alginic acid and chitosan, and porous inorganic carriers such as celite, ceramics, activated carbon, etc., are used for adhesion or immobilization of cells for such applications.

For example, Korean Patent Publication No. 1999-021170 discloses a step of using a gelling solution after heat treatment and drying of a porous inorganic carrier, followed by addition of a cell suspension and an organic polymer solution to form a polymer membrane on the surface of the porous inorganic carrier. Disclosed are a method for immobilizing a cell containing the same.

The conventional method of immobilization as described above is to allow cells to adhere to the carrier while naturally proliferating in the pores of the carrier, so that the slow growth rate of the cells takes long time to immobilize the cells. In addition, there is a disadvantage that the carrier surface area is wasted because the cells do not adhere to all surfaces of the carrier.

Therefore, in order to induce cell adhesion, efforts have been made to improve cell adhesion by changing the functional groups on the surface of the solid granular product by using covalent bonds, but there are difficulties in working. In addition, as a method of gelling organic polymers to porous inorganic carriers, only porous inorganic carriers such as celite, ceramics, and activated carbon are used, but polyethylene, polypropylene (PE) Examples of using polymer resins such as PP), polyvinyl chloride (PVC), silicone, and the like are extremely rare.

On the other hand, the polymer resin is not suitable for cell growth and poor adhesion to the cell, because the surface of the polymer resin is hydrophobic rather than hydrophilic property, which is the basis for cell adhesion or blood adhesion. In order to improve this, in recent years, researches to improve cell adhesion by modifying the polymer resin surface have been actively conducted.

The typical method is a method of grafting with a monomer synthesis method, but this method has a problem that the synthesis process is complicated and difficult to control the desired surface, and there is also a plasma surface treatment method using ammonia gas. However, this is a problem that the functional groups remaining on the surface gradually decreases over time.

In addition, Korean Patent Publication No. 2003-026943 discloses a cell immobilization carrier having an alginic acid film adhered on a film made of polyolefin, PVC, or derivatives thereof. After making the surface have hydrophilic properties, the method is applied to fix the cells by applying an alginate gel thereon.

However, the plasma treatment disclosed in the patent document is a continuous plasma treatment method, and has a problem in that the survival time of radicals is short, so that the reaction efficiency is low, and control precision for a suitable surface structure is somewhat inferior.

In addition, the method of attaching the amine group to the surface in order to improve the suitability of the cell is known to be important for the adhesion of the cell, the carrier to which the alginic acid gel is applied has a disadvantage that is limited in its use range.

Therefore, the study of developing a polymer resin having excellent cell adhesion by modifying the surface of the polymer resin such as polystyrene and polyethylene terephthalate and using the amine-based material as a precursor by pulsed plasma polymerization that has improved the disadvantage of continuous plasma polymerization. Necessity arose constantly.

Thus, the present inventors solve the problems of the continuous plasma treatment method of the prior art as described above, when surface treatment by pulsed plasma polymerization using an allylamine monomer to attach the amine group to the surface, excellent cell adhesion In addition, the survival time of radicals was long, and it was confirmed that a carrier for cell adhesion can be obtained with high reaction efficiency and more precise control of a suitable surface structure, thereby completing the present invention.

After all, the main object of the present invention is to provide a surface treatment method of a polymer resin that maximizes cell adhesion using pulsed plasma.

Another object of the present invention is to provide a cell adhesion carrier prepared by using the surface treatment method of the polymer resin, which can control growth by adhering cells.

The present invention provides a method for surface treatment of polymer resin by pulsed plasma polymerization using an amine monomer while maintaining a pulse frequency of 1000 Hz or less and a duty cycle of 100% or less.

In this case, the amine-based monomer is not particularly limited thereto, but is preferably allylamine, and the polymer resin is not particularly limited thereto, but may be polystyrene, polypropylene, polyethylene terephthalate, polyvinyl chloride, and polyolefin. It is preferably at least one resin selected from the group consisting of.

In addition, the present invention provides a cell adhesion carrier for controlling the adhesion and growth of cells using a polymer resin modified surface by the surface treatment method of the polymer resin.

The pulse frequency used in the present invention is preferably 200 to 1,000 Hz, more preferably 250 to 350 Hz.

In the present invention, the carrier means a concept including a cell culture petri dish or a plastic case of a hemodialyzer.

Plasma polymerization (plasma polymerization or plasma enhanced chemical vapor deposition) means that gaseous monomers of organic and organic metals are synthesized (deposited) in the form of a thin film or powder having a very high crosslinking density on the surface of the substrate in a plasma discharge state. Occurs in the presence of monomers of chemicals containing atoms that form chains such as silicon, silicon, and the like. In most cases, monomer molecules introduced into a plasma state are broken down into activating particles by plasma energy, so that only a partial chemical structure of the injected monomer is preserved so as to have a crosslinked and irregular structure.

The structure and properties of composites can be precisely controlled by plasma process factors such as pressure, gas input, gas type, voltage, voltage bias, etc. Gradients that gradually change physical properties in the thickness direction using these properties Layer structures can be produced.

In general, a method of generating a plasma is generated by an electric field. Plasma can be divided into direct current glow discharge, alternating current (50-60 Hz) plasma, audio (kHz) -radio (MHz) frequency plasma and microwave plasma (GHz) discharge, depending on the power source and frequency band used.

Methane, ethane, ethylene, acetylene, benzene and the like are widely used hydrocarbon raw materials for synthesizing hydrogenated carbon films, and the resulting plasma polymers have improved microhardness, refractive index, abrasion resistance, and the like.

Research into plasma polymers polymerized using organosilicon, halocarbon, and carbon containing metals as raw materials has been actively conducted.

Plasma polymers produced using organosilicon monomers exhibit good thermal, chemical stability, good electrical, optical and biocompatible properties.

Mostly studied organosilicon monomers include silane, disiloxane (SiOSi), disilazane (SiNHSi), and disilthiane (SiSSi).

In the plasma polymerization reaction, monomers are often introduced before the glow discharge, and the glow discharge is usually formed by the electric field under a low pressure of 10 Torr or less.

Fragments of atoms, radicals, and ions in contact with the plasma have a strong surface and activation. Although the degree of ionization is as low as 10 ^-5 to 10 ^-7 , charged particles are very important for determining the deposition rate and the chemical structure of the polymerization product.

The electrons create a non-equilibrium or "cold" plasma that is not in thermodynamic equilibrium with the gas molecules, and as a result, the temperature of the gas molecules is similar to room temperature and no degradation of reactants or products occurs. Applicable

Due to the plasma off time, the pulsed plasma polymerization technology is an important alternative to overcome the shortcomings of polymer materials because the heat of the sample does not rise during the surface treatment process. It was used as a method of improving the cell adhesion of the polymer resin of the present invention because it can be applied without being excellent and not only excellent adhesion but also can process a three-dimensional substrate.

In the present invention, pulsed plasma polymerization was performed for 3 minutes. Polyethylene terephthalate (PET), a polymer resin used, was used as a 3M pp2910 film, and its thickness was 50 μm. Allylamine (allylamine) used as a monomer in this experiment used a reagent grade of Aldrich's 99% purity, and the basic physical properties of the allylamine used are shown in Table 1 below.

Based on the previous experimental results, 50 watt or less of the range which does not damage the surface of the polymer was selected as the basic treatment plasma power.

When the plasma power was 50 watts or more, the surface properties of the polymer material were deteriorated and the polymer film was distorted, and the color was changed from transparent to yellow due to the surface deterioration.

In the present invention, the allylamine monomer was stably and quantitatively introduced into the plasma reactor using a liquid vaporizer, and the plasma characteristics during the plasma polymerization reaction were diagnosed.

pulse plasma  Polymerization experiment

Contact angle and cell adhesion experiments were performed on samples made by pulsed plasma polymerization controlling the duty cycle.

Oxygen plasma treatment was performed prior to pulsed plasma polymerization. The duty cycle was adjusted to 100%, 50% and 30% at a frequency of 300 Hz to determine the effect of the pulse. By varying the duty cycle, it was possible to have a wide range of contact angles.

This meant that the surface energy could be adjusted as needed by the pulse experiment. In the case of Example 1 (duty cycle: 50%, power: 10 watts, allylamine: 2 g / hr) of the experiment, the contact angle was relatively high (41 °) and showed the best cell adhesion.

Example 1 showed more excellent cell adhesion than that obtained by the RF plasma (Comparative Example 4, Comparative Example 5), confirming the possibility of improving cell adhesion by the duty cycle control. In the present invention, the polymerization rate was about 20 nm / min, and the contact angle was lowered by 26 to 42 degrees compared to 78 degrees of untreated PET.

Cell inoculation in this experiment was carried out under the following conditions, the cell culture time was measured after 1 day, 2 days, 4 days, Table 2 below shows only the number of cells after 4 days.

Cell inoculation condition

Cell line: Fibroblast

Medium: RPM I (Fibroblast) with 10% FBS

Culture conditions: 37 ℃, 5% CO ₂

Inoculation Density: 4 * 10 ⁴ cells / cm ²

Cell incubation time: 1-4 days

analysis

Cell culture was carried out using a polymer film whose surface was modified by plasma polymerization, and cell culture was made by cutting the film into a circular shape and equilibrating with PBS. After 1 hour, 2 days, and 4 days, the cell number was counted and biocompatibility was confirmed by a hemocytometer and MTT test.

In addition, the present invention may include various monomer derivatives. In the present invention, any amine may be used, but allylamine is most preferred.

The surface treatment method of the present invention and the polymeric medical device are not limited in shape, but the plate-like form is most preferred.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example

In this example and the comparative example, the allylamine input amount was 2 g / hr and an RF generator (13.56 MHz) was used. The pressure in the chamber was kept below 20 mTorr before the pulsed plasma polymerization, vaporized monomer was added thereto, and the pulsed plasma polymerization was carried out. The process pressure was 250 mTorr.

Example  One :

The mixture was treated for 3 minutes by plasma polymerization, and polyethylene terephthalate was used as a 329 pp2910 film. The thickness was 50 μm. Specific experimental methods are shown in Table 2 below.

Example  2 to 4 :

In the same manner as in Example 1, but the frequency and duty cycle was adjusted.

Comparative example  1 to 5 :

Comparative Example 1 is an untreated sample, which is a result of only washing with alcohol and then drying the sample without any other treatment. Comparative Example 5 in Comparative Example 2 is as shown in Table 2.

Example  5 :

Polystyrene resin (150 µm film) was used instead of polyethylene terephthalate.

Table 2 shows the experimental conditions and results of the Examples and Comparative Examples, as shown in Table 2, the contact angle is low in all cases by the plasma treatment, especially the duty cycle is reduced under the same conditions Accordingly, it can be seen that as the plasma ON time decreases, the contact angle becomes higher. Comparing the results of Table 2, even though the contact angles are similar in size, the cell adhesion shows a difference, and even when the contact angle is large, it can be seen that the cell adhesion is almost similar. Therefore, the correlation between contact angle and cell adhesion was low.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a surface treatment method of modifying the surface of a polymer resin using a pulsed plasma and an amine-based material as a precursor, thereby providing a surface having excellent cell adhesion. It can be widely used in biomaterial product development.

Claims (5)

A method of surface treatment of a polymer resin by a pulsed plasma polymerization method using an amine monomer while maintaining a 10 watt pulse frequency of 300 Hz and a duty cycle of 30 to 60%. The method of claim 1, The amine monomer is an allylamine (allylamine) surface treatment method of a polymer resin, characterized in that. The method of claim 1, The polymer resin is a surface treatment method of a polymer resin, characterized in that at least one resin selected from the group consisting of polystyrene, polypropylene, polyethylene terephthalate, polyvinyl chloride and polyolefin. delete Cell adhesion carrier for controlling the adhesion and growth of cells comprising a polymer resin surface-modified by the surface treatment method of claim 1.