KR101091872B1 - Polymer Mutlilayer Coatings including Cationic pH-Sensitive Polymer as Biomaterial and their synthesis - Google Patents

Abstract

The present invention relates to a polymer multilayer membrane for a biomaterial having reduced cytotoxicity by alternately stacking a cationic polymer layer and an anionic polymer layer using a pH sensitive polymer of Formula 1 below.

[Formula 1]

(Wherein, R is any one selected from imidazole, (C1-C20) alkylamine, (C2-C20) arylamine, pyridine, (C2-C20) aromatic amine.)

pH sensitive polymer, cationic polymer layer, anionic polymer layer, cytotoxicity, layer-by-layer

Description

Polymer Mutlilayer Coatings including Cationic pH-Sensitive Polymer as Biomaterial and their synthesis}

The present invention relates to a polymer multilayer membrane that can be used in biomaterials, by alternately stacking a cationic pH sensitive polymer layer and an anionic polymer layer, a polymer that can be safely applied to the human body by reducing the cytotoxicity of the cationic polymer A multilayer film and a method for manufacturing the same.

In biocompatible polymers, polymers used to replace parts of the body and various medical activities including diagnosis and treatment are used. Recently, biodegradable polymers or amphiphilic polymers having both hydrophobic and hydrophilic groups are used to change the structure of molecules. In addition, studies on drug carriers that control sol gel transition by forming hydrogels with block copolymers have been actively conducted.

Among these, research on pH-sensitive polymers is being actively conducted, and these pH-sensitive polymers form aggregates at specific pH, and when the pH-sensitive polymers are applied to medical materials and biomaterials as they are, the amount of aggregates increases. Cell proliferation did not progress smoothly and was shown to be cytotoxic.

The present invention is to provide a method for use as a polymer for biomaterials by reducing the cytotoxicity of pH-sensitive polymers.

Specifically, the present invention is to provide a method for producing a polymer multilayer membrane using a pH sensitive polymer.

The present invention is to reduce the cytotoxicity of the pH-sensitive polymer in order to reduce the variation or specific toxicity of the cell activity occurs in a specific pH-sensitive polymer, when the pH-sensitive polymer is a cationic polymer by alternately stacked with an anionic polymer It relates to a polymer multilayer membrane characterized by reducing cytotoxicity.

The present inventors completed the present invention by manufacturing a multilayer electrolyte membrane and developing a technology capable of using the multilayer electrolyte membrane by alternately stacking a positive and negatively charged polymer solution to a variety of hydrogen ion concentration (pH) on a substrate such as glass or silicon membrane It was.

Specifically, the present invention relates to a polymer multilayer membrane for biomaterials having a reduced cytotoxicity by alternately stacking a pH-sensitive copolymer layer and an anionic polymer layer using a cationic polymer of Formula 1 below.

[Formula 1]

(Wherein, R is any one selected from imidazole, (C1-C20) alkylamine, (C2-C20) arylamine, pyridine, (C2-C20) aromatic amine.)

In addition, in the present invention, the anionic polymer uses any one selected from poly (meth) acrylic acid, poly (eta) acrylic acid, polybenzylsulfonic acid, hyaluronic acid, polyaspartic acid, and the like.

Next, a method for producing the polymer multilayer film of the present invention will be described.

a) cleaning the substrate;

b) alternately applying a cationic polymer solution and an anionic polymer solution using the pH sensitive polymer of Formula 1 to the cleaned substrate;

[Formula 1]

(Wherein, R is any one selected from imidazole, (C1-C20) alkylamine, (C2-C20) arylamine, pyridine, (C2-C20) aromatic amine.)

c) repeating step b) one or more times to produce a polymer multilayer film;

It includes.

In addition, in the present invention, in the step c), the outermost portion of the polymer multilayer film is preferably coated with a cationic polymer aqueous solution.

The pH of the cationic polymer solution and the anionic polymer solution is preferably 2 to 10, more preferably 3 to 7.

Hereinafter, the present invention will be described in more detail.

The pH-sensitive polymer synthesis structure used in the present invention is represented by the following formula (1).

[Formula 1]

(Wherein, R is any one selected from imidazole, (C1-C20) alkylamine, (C2-C20) arylamine, pyridine, (C2-C20) aromatic amine.)

Although not limited, the weight average molecular weight of the polymer of Chemical Formula 1 is preferably 10000 to 200000.

In the present invention, the pH-sensitive polymer is preferably used by purification by precipitation (DMF / acetone), and when such purification is carried out, the residual monomer is removed, and the molecular weight distribution is also lowered, so that the sample exhibits a solubility transition according to pH. Can be obtained, further reducing the cytotoxicity. As a method of purifying the polymer, the precipitation method is to dissolve the polymer in a dimethylformamide (DMF) solution, and then immerse it in acetone. It is also possible to purify purified water by dialysis using a cellulose ester (Spectrum, MWCO = 500-1000) separator.

Purified polymer is used to make an aqueous solution using deionized water (> 18MΩcm Millipore Milli-Q), the aqueous solution is prepared in a concentration of 0.01M ~ 0.1M. In order to adjust the pH conditions, it is adjusted using 0.1 M HCl, NaOH aqueous solution. In this case, the pH of the cationic polymer solution and the anionic polymer solution is preferably 3 to 7. Since the bond between the cationic polymer and the anionic polymer is well made in the pH range, it is possible to reduce the cytotoxicity of the cationic polymer.

The polymer multilayer membrane is manufactured by alternately adsorbing at various pH conditions using opposite charges of the polymer of Formula 1, which is a positively charged polymer, and polyacrylic acid, a negatively charged polymer.

That is, the present invention manufactures a polymer multilayer film in the following steps.

a) cleaning the substrate;

b) alternately applying a cationic high molecular aqueous solution and an anionic aqueous polymer solution using a pH-sensitive polymer of Formula 1 to the cleaned substrate;

[Formula 1]

(Wherein, R is any one selected from imidazole, (C1-C20) alkylamine, (C2-C20) arylamine, pyridine, (C2-C20) aromatic amine.)

c) repeating step b) one or more times to produce a polymer multilayer film;

It includes.

First, in the step a), it is preferable to wash the substrate (glass or silicon slide) to form a coating film with dilute detergent solution, and then several times with deionized water, and then dry with nitrogen gas. It is not a big limitation.

In the next step b), the polymer multilayer film has a weak negative or positive charge on the surface of the polymer, so that the charge of the polymer electrolyte coated on the substrate is better in adhesion than the possible charge of the substrate. It is not limited. For example, in the case of a glass substrate, since the surface of the substrate has a weak negative charge, it is immersed in a positively charged polymer, for example, an aqueous solution of a pH sensitive polymer of Formula 1 (hereinafter referred to as C10-IM50) for a sufficient time. After washing, it is dried using nitrogen gas. Again, it is sufficiently immersed in an aqueous solution of polyacrylic acid (PAA) or polymethacrylic acid (PMA), which is a negatively charged polymer, and through the same washing process, a coating film in which the two polymer electrolyte layers are combined is formed. The coating film formed in this process is called 1 bilayer (denoted as 1BL), and the process is repeated until the desired thickness is formed. In the case of the above-described polymer, the polymer multilayer film formed may be referred to as C10-IM50 / PAA and C10-IM50 / PMA multilayer film. After the polymer multilayer film is finished, it is quickly dried using nitrogen gas.

The positively charged or negatively charged polymer electrolyte component used in the present invention is not particularly limited as long as it is a component that is commonly used. For example, C10-IM50, PAA, or PMA as mentioned above may be mentioned.

The polymer multilayer membrane prepared according to the present invention can provide a material suitable for use in biomaterials and medical fields because the cytotoxicity of the pH sensitive polymer is reduced.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the present invention is not limited to the following examples.

[Example]

(1) purification and sample solution preparation

The polymer used in the experiment was a polysuccinimde copolymer containing imidazole (C10-IM50, Mw = 40000) synthesized by Aekyung Industrial Co., Ltd. The pH-sensitive polymer synthesis structure is shown in the following formula (2).

[Formula 2]

The synthesized pH-sensitive polymer was used in the experiment after purification of the precipitation method (DMF / acetone) which is dissolved in dimethylformamide and then precipitated in acetone. The distribution was also 10 times narrower, yielding a white powder.

The purified polymer was used to make an aqueous solution using deionized water (> 18MΩcm Millipore Milli-Q), and was adjusted using HCl and NaOH aqueous solution (0.1M) to meet pH conditions.

1) Solubility Changes According to pH of Synthetic pH-sensitive Polymers

In order to observe the solubility change according to the pH of the synthesized pH-sensitive polymer was added 0.05g of pH-sensitive polymer in 50ml of deionized water and stirred for about 12 hours to prepare an aqueous solution. The solubility change was measured using a visible-ultraviolet spectrometer while gradually increasing the pH of the aqueous solution from 3.0 to 9.0 with 0.1 M HCl and NaOH. The results are shown in Figure 1, it was confirmed that the transition of solubility occurs at pH6 ~ pH8.

2) Hydrophobic material loading experiment of pH sensitive polymer

6-carboxyfluorescein-N-hydroxy succinimide ester (Sigma Co., Ltd.) of the following Chemical Formula 3 was used as a loading test of the hydrophobic material using the pH sensitive polymer.

(3)

0.05 g of pH sensitive polymer was added to 50 ml of deionized water, and stirred for about 12 hours to prepare an aqueous solution. In order to mount the fluorescent material in the pH-sensitive polymer (C10-IM50) aqueous solution, the fluorescent material was prepared in an aqueous solution at a concentration of 0.1 micromole, and then mixed with the aqueous polymer solution in a 1: 1 volume ratio to prepare a mixed solution. The mixed solution was used to remove the fluorescent material not bound by dialysis in deionized water for 24 hours using a molecular weight cut-off 1000 membrane, and then measured the degree of mounting on the polymer while adjusting the pH of the mixed solution. The results are shown in Figure 2, it can be seen that the most amount can be mounted in the vicinity of pH 7, the polymer aggregates are formed.

(2) Preparation and analysis of polymer thin film by layer-by-layer method

C10-IM50, a cationic polymer, and poly (acrylic acid), (PAA), an anionic polymer, were combined to form a polymer multilayer. PAA used a Polysciences reagent with a molecular weight (Mw = 90,000). The process of making and washing the aqueous solution consisted of deionized water (> 18 MΩcm Millipore Milli-Q). C10-IM50 and PAA aqueous solution were made of 0.01M. The pH of the solution was adjusted to 0.1 M each of HCl and NaOH.

The glass slide used as a substrate was put together with a detergent in a washing bottle, washed for 15 minutes using an ultrasonic cleaner, and then washed three times with deionized water and dried using nitrogen gas. The washed substrate was immersed in a C10-IM50 cationic aqueous solution for 20 minutes, then rinsed twice, and then immersed in an anionic polyelectrolyte aqueous solution PAA for 15 minutes and then rinsed twice with deionized water for 2 minutes. This process is called 1 bilayer and the number of bilayers is increased by the desired thickness. After the film was finished, it was quickly dried using nitrogen gas.

In the same manner as above, the C10-IM50 / PAA multilayer film was prepared at pH3.0 / 3.0 and pH5.0 / 5.0, respectively. In addition, the multilayer film of each pH condition was prepared by 2, 2.5, 4, 4.5, 6, 6.5, 8, 8.5, 10, 10.5 bilayer to prepare a film.

1) Check the lamination of the film

Film lamination confirmation was measured by using a methylene blue (Methylene blue) aqueous solution capable of binding to the PAA. Each of the multilayer films prepared above was soaked in 1mM Methylene blue aqueous solution at pH 7.4 for 15 minutes, rinsed twice with deionized water for 2 minutes, and dried over nitrogen gas. And absorbance was measured using UV-Visible spectroscopy. The results are shown in FIG. 3 (condition of pH 3) and FIG. 4 (condition of pH 5). As shown in the figure, as the formation of the multilayer film increases, the contained dye increased, indicating that the multilayer film was well formed under these conditions.

2) Wetting test of film surface

In the wettability test of the film surface, the contact angle of water was measured by using CA-A Contact angle analyzer of Face Company. Deionized water was used for the contact angle measurement, and 15 ml of droplets were formed by using a syringe, and then the measurement plate on which the film was lifted was gradually raised to contact the droplets so that there was no influence of gravity on the contact angle measurement.

Contact angle is the angle between the solid surface and the liquid surface when the liquid is thermodynamically balanced on the solid surface. At this time, since the angle varies depending on the state of the solid surface and the type of liquid, the contact angle shows the surface energy, the uniformity of the surface, and the degree of hydrophilicity. The contact angle was measured to evaluate the wettability of the films formed under both pH conditions. The measurement results are shown in FIG. 5 (condition of pH 3) and FIG. 6 (condition of pH 5). As a result of wettability (wetability) test on the film having the surface of C10-IM50 and the film having the surface of PAA, it was found that the film having the outermost surface of C10-IM50 exhibited more hydrophobic properties than the surface of PAA. there was.

3) Biological Stability Test of Multilayer Membrane Film between Cationic Polymer Copolymer and Anionic Polymer

Cell culture experiments were carried out to determine the biological stability and nontoxicity of the cationic polymer copolymer (C10-IM50) itself used in this experiment, the agglomerates of the anionic polymer that can be combined with the polymer, and the polymer multilayer membrane formed of a thin film. . As a substrate for experiments on the adsorption and proliferation of cells, a 60 mm diameter cell culture polystyrene dish (TCPS Tissue Culture Grade Polystyrene Dish) was used. For the experiment, the cells were observed using a culture medium and incubated under appropriate conditions (37 ° C., 5% CO ₂ ). For cell experiments, samples were used after sterilization with 70% ethanol and dried in a clean bench. After the cells were added to the sample, the adsorption process of the cells was observed for 1, 3 and 5 days.

Renal cells (HEK 293 cells) were used as a species of cells useful in the medical field and biomaterials because of their rapid proliferation and easy cultivation.

3-1. Cell Stability Test of Aggregates between Cationic Copolymers and Anionic Polymers

First of all, for the cell stability test of the cationic copolymer (C10-IM50) itself, we examined the effect on the cell culture by adding C10-IM50, a pH sensitive polymer that forms aggregates at pH 7.4, to the cell culture sample. In the case of using the concentration of 0.01M C10-IM50 used in the previous experiment, the amount of aggregates was too large, and diluted again to 20% and mixed with the cell culture solution 1: 1 to culture the cells (sample A in the table below). Figure 7 (a) is a micrograph of the results of observing the degree of proliferation for three days in the cell culture solution, sample A solution, containing the C10-IM50 solution (Scale bar, 50 mm) compared to normal cell proliferation C10 When -IM50 was used, initial adsorption on day 1 occurred to some extent, but proliferation was significantly reduced when observed on day 3, which was considered to be toxic to cationic polymers.

In addition, using the polyacrylic acid (PAA), an anionic polymer capable of binding to C10-IM50 used in this experiment, polymer aggregates were formed and cell culture experiments were performed to know the biological stability and nontoxicity of the aggregates. . For the experiment, the cationic copolymer (C10-IM50) and the anionic polymer (PAA) were mixed at the ratio of sample B ~ D in Table 1 below to form aggregates and added to the cell culture solution to culture the cells in the mixed culture solution. The adsorption and proliferation of cells were observed for 1 and 3 days. FIG. 7B shows the result of sample B, FIG. 7C shows the sample c, and FIG. 7D shows the result of sample D. FIG.

TABLE 1

In cell culture experiments, cell proliferation was more active as the amount of PAA in the aggregates increased than when only the cationic copolymer was present.

3-2. Cell Stability Test in Polymer Electrolyte Multilayer Membrane Films

Substrate for experiments on the adsorption and proliferation of the cells is a 60mm diameter cell culture polystyrene dish (TCPS Tissue Culture Grade Polystyrene Dish) to coat 2/3 of this surface with a multilayer thin film in the same manner as in Example 1 The coated and uncoated parts (Bare) can be compared and observed. The coating films of 10 and 10.5 bilayr were observed for both the C10-IM50 outermost film and the PAA outermost film. For the experiment, the cells were observed using a culture medium and incubated under appropriate conditions (37 ° C., 5% CO ₂ ). For cell experiments, samples were used after sterilization with 70% ethanol and dried in a clean bench. After the cells were added to the sample, the adsorption process of the cells was observed for 1, 3 and 5 days.

To determine the interaction between C10-IM50 (pH 3.0) / PAA (pH 3.0) and C10-IM50 (pH 5.0) / PAA (pH 5.0) multilayer thin film, we use kidney cells (HEK 293 kidney cells). The experiment was carried out. The results are shown in FIGS. 8 and 9. As shown in FIGS. 8 and 9, in the C10-IM50 (pH 3.0) / PAA (pH 3.0) conditions, the adsorption of cells occurs very well, similar to the case where the coating is not performed, and the C10-IM50 (pH 5.0) / PAA (pH 5.0). ) Shows increased cell adsorption than only C10-IM50 aggregates were introduced. In these cases, cationic polymers are generally known to be highly toxic under conditions of high ionization, and they appear to have high biological stability in structures that are well bonded to relative polymers, that is, anionic polymers.

 In the case of both pH membrane films, there was a difference in cell adsorption, but there was no change in cell activity or specific toxicity.

1 is a change in solubility according to the pH of purified C10-IM50

2 is an absorbance showing the mount retention of the hydrophobic fluorescent substance of the C10-IM50 polymer according to pH

3 is a graph showing the results of increasing the dye content according to the increase in the thickness of the polymer multilayer film prepared under the pH 3 condition

4 is a graph showing the results of increasing the dye content according to the increase in the thickness of the polymer multilayer film prepared under the condition of pH 5

Figure 5 is a graph measuring the contact angle to evaluate the wettability (Wettability) of the polymer multilayer film prepared at the pH 3 conditions

Figure 6 is a graph measuring the contact angle to evaluate the wettability (Wettability) of the polymer multilayer film prepared under the condition of pH 5

Figure 7 is a micrograph showing HEK 293 cell interaction of purified C10-IM50 with PAA aggregates. (a) is a photograph showing the adsorption and proliferation of cells for 1 to 3 days when pure C10-IM50 aggregates were included in the cell culture medium (1: 1 mixture by volume and culture volume), and (b) is C10-IM50 80. Cell experiments in which% and PAA 20% aggregates were included in the cell culture medium, (c) shows that 50% C10-IM50 and 50% PAA aggregates were included in the cell culture medium, and (d) showed the highest amount of PAA. It is observed that the adsorption and proliferation of cells for 1 ~ 3 days when the aggregates of 20% of C10-IM50 and 80% of PAA were included in the cell culture medium (scale bar, 50㎛). The more proliferation of the cells, the more active the results.

FIG. 8 is a micrograph showing HEK 293 cell interaction on C10-IM50 (3.0) / PAA (3.0) multilayer thin film (Scale bar, 50 μm)

9 is a micrograph showing HEK 293 cell interaction on a C10-IM50 (5.0) / PAA (5.0) multilayer thin film (Scale bar, 50 μm).

Claims (6)

A polymer multilayer membrane for a biomaterial having reduced cytotoxicity by alternately stacking a cationic polymer layer using a pH-sensitive polymer of Formula 2 and an anionic polymer layer using polyacrylic acid. [Formula 2]

delete a) cleaning the substrate; b) alternately applying a cationic polymer aqueous solution containing a pH sensitive polymer of Formula 2 and an anionic aqueous solution containing polyacrylic acid to the cleaned substrate; [Formula 2]

c) repeating step b) one or more times to produce a polymer multilayer film; Method for producing a polymer multilayer film for a biological material comprising a. The method of claim 3, Method of manufacturing a polymer multilayer film for a biomaterial, characterized in that repeating the step of alternately stacked in step c) 2 to 10 times. The method of claim 4, wherein PH of the cationic polymer solution and anionic polymer solution is 3 ~ 7 method for producing a polymer multilayer membrane for a biomaterial. delete

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