JP5835769B2 - Hydrophilic porous membrane and method for producing the same, medical anti-adhesion membrane and cell growth substrate - Google Patents

Description

  The present invention relates to a hydrophilic porous membrane that can be suitably used in the medical field and the like.

  Biodegradable polymers such as polylactic acid are expected to be applied to medical fields such as hemostatic materials, adhesion / adhesion prevention materials, and regenerative engineering materials.

  However, polylactic acid itself is a hard material, and as such, application to the medical field is limited. In order to obtain a material that can be widely applied in the medical field, for example, polylactic acid may be a copolymer with other polymer. Specifically, a copolymer of polylactic acid and polyglycolic acid has been put into practical use as a surgical thread that does not require a thread removal. Alternatively, as disclosed in Patent Document 1, a multi-block copolymer of polylactic acid and polycaprolactone has excellent mechanical properties and the like, and is used as a medical material such as a medical matrix and a wound coating material. It is a suitable material.

Japanese Patent No. 3526856

  However, as a result of diligent research by the present inventors, the multi-block copolymer of polylactic acid and polycaprolactone itself is hydrophobic and is not suitable for medical use as it is. It has been found that even if the surface of a molded product of a multi-block copolymer (such as a membrane) is made hydrophilic, the molded product easily moves in the laboratory animal (rat) body simply by making it hydrophilic. That is, the adhesion to the wet surface was not sufficient. For example, even when the multiblock copolymer is installed as an adhesion preventing film in an affected area in a living body, it has been found that the adhesion preventing film moves from the affected area and the affected area adheres with time. Further, the method disclosed in Patent Document 1 has also been found to make it difficult to synthesize a multiblock copolymer of polylactic acid and polycaprolactone with good reproducibility.

  Then, this invention makes it a subject to provide the hydrophilic porous membrane which was excellent in the adhesiveness with respect to a wet surface while having biodegradability.

As a result of intensive studies by the present inventors, the following findings were obtained.
(1) When a multi-block copolymer of polylactic acid and polycaprolactone is formed and subjected to alkali hydrolysis, voids communicating from the front surface to the back surface can be generated in the film. That is, a porous film can be obtained.
(2) The voids formed by alkali hydrolysis have their inner wall surfaces also made hydrophilic. That is, it is possible to hydrophilize not only the surface of the film but also the inside by the gap communicating from the front surface to the back surface. Such a membrane can be soaked with water. Moreover, water can permeate | transmit from the surface to a back surface by making a space | gap communicate from the surface to the back surface.
(3) A porous membrane that has been hydrophilicized to the inside has excellent adhesion to wet surfaces. That is, it is physically in close contact with the affected area surface in the living body and does not move easily.
(4) A porous membrane produced from a multi-block copolymer of polylactic acid and polycaprolactone is easily decomposed in vivo.
(5) Cell culture is possible on a porous membrane prepared from a multi-block copolymer of polylactic acid and polycaprolactone, and the cells penetrate into the membrane.
(6) In order to efficiently produce a multi-block copolymer of polylactic acid and polycaprolactone, first, a diblock copolymer (oligomer) of polylactic acid and polycaprolactone is synthesized, and then the diblock is produced. The copolymer may be converted into a multiblock copolymer by self-polymerization.

The present invention has been made based on the above findings. That is,
The first aspect of the present invention is a hydrophilic porous membrane comprising a multi-block copolymer of polylactic acid and polycaprolactone, and having voids formed by alkali hydrolysis treatment.

  In the first aspect of the present invention, the thickness of the film is preferably 5 μm or more and 100 μm or less. Thinner films have insufficient film strength and are difficult to use alone. However, it is possible to use the film according to the present invention in an overlapping manner, and a film having a thickness of 100 to 1000 μm or more can be obtained by overlapping several films.

  The second aspect of the present invention is a hydrophilic porous membrane comprising a multi-block copolymer of polylactic acid and polycaprolactone and having voids communicating from the membrane surface to the membrane back surface.

In 2nd this invention, it is preferable to have said space | gap by carrying out the alkaline hydrolysis process. In such a film, in particular, when water droplets are added to the film surface, the phenomenon of the water droplets soaking into the film is noticeably observed.
In order to generate “a void communicating from the membrane surface to the membrane back surface” by the alkali hydrolysis treatment, for example, a contact angle with water of 20 to 30 ° and a membrane having a hydrophilic surface are further subjected to alkaline hydrolysis. By performing the treatment, the gap can be communicated from the film surface to the film back surface.

  Whether or not voids communicate from the film surface to the film back surface can be determined by observing the cross section of the film with an electron microscope or the like. Alternatively, a simple determination can be made by placing a water droplet on the surface of the membrane and confirming whether or not the water has penetrated.

  In the second aspect of the present invention, the thickness of the film is preferably 5 μm or more and 100 μm or less. Thinner films have insufficient film strength and are difficult to use alone. However, it is possible to use the film according to the present invention in an overlapping manner, and a film having a thickness of 100 to 1000 μm or more can be obtained by overlapping several films.

  The third aspect of the present invention is a medical adhesion prevention film using the hydrophilic porous membrane according to the first aspect of the present invention or the second aspect of the present invention.

  4th this invention is the base material for cell proliferation which uses the hydrophilic porous membrane which concerns on 1st this invention or 2nd this invention.

  The fifth aspect of the present invention includes a step of obtaining a multiblock copolymer of polylactic acid and polycaprolactone, a step of forming the obtained multiblock copolymer into a film shape, and alkaline hydrolysis of the formed membrane. And a step of producing a void in the membrane by processing.

  In the fifth aspect of the present invention, it is preferable to perform an alkali hydrolysis treatment with an alkali having a pH of 12 or more.

  In the fifth aspect of the present invention, the thickness of the film is preferably 5 μm or more and 100 μm or less.

  In the fifth aspect of the present invention, it is preferable that the alkali hydrolysis treatment is performed until the voids are communicated from the front surface to the back surface of the membrane.

  In the step of obtaining a multiblock copolymer according to the fifth aspect of the present invention, a multiblock copolymer is prepared by synthesizing a diblock copolymer of polylactic acid and polycaprolactone and subjecting the diblock copolymer to post-polymerization. It is good to obtain a unity.

In the present invention, the “hydrophilic porous membrane” means a membrane through which water can permeate and permeate from the membrane surface to the membrane back surface. In the case of a lipophilic porous membrane, even if it is a porous membrane, water stops on the membrane surface, and water cannot permeate and permeate from the membrane surface to the membrane back surface.
In addition, the molded object which consists of a polylactic acid-polycaprolactone multiblock copolymer has the surface where the contact angle with respect to water will be about 70 degrees in the case where a hydrophilic treatment is not carried out. Since the surface is made hydrophilic, the surface has a contact angle with water of 40 ° or less, preferably 30 ° or less, more preferably 25 ° or less. If the hydrolysis treatment is further advanced from this state and the treatment is stopped in an appropriate time, a void communicating from the membrane surface to the membrane back surface can be formed, resulting in a hydrophilic porous membrane into which water permeates. In addition, the water permeability improves when treated for a long time, but when it is too long, the strength of the membrane decreases. The method for setting the contact angle of water on the membrane surface to a desired range is not limited to the above, and may be made hydrophilic by, for example, ozone treatment.

  According to the present invention, using a multi-block copolymer of polylactic acid and polycaprolactone, a hydrophilic porous membrane capable of absorbing and transmitting water from the front surface to the back surface can be obtained. The hydrophilic porous membrane has biodegradability and excellent adhesion to a wet surface. For example, it can be held in a predetermined position for a long period of time without being displaced in the living body, and is appropriately decomposed in the living body.

It is a figure for demonstrating the synthetic pathway of a polylactic acid-polycaprolactone multiblock copolymer. It is a SEM image figure which shows the surface state of the evaluation film | membrane used in the Example. It is a photograph figure which shows the state after 6-day progress, when a hydrophilic porous membrane is installed in the affected part surface of a rat liver. (A) is the photograph figure which showed only the state of the cell after performing cell culture on a hydrophilic porous membrane, (B) is the synthetic photograph figure which showed the state of both a film | membrane and a cell.

<Hydrophilic porous membrane>
(1. First embodiment)
The hydrophilic porous membrane of the present invention according to the first embodiment comprises a multi-block copolymer of polylactic acid and polycaprolactone, and has a void formed by alkali hydrolysis treatment. It is.

(1.1. Multiblock copolymer of polylactic acid and polycaprolactone)
In the present invention, as the multiblock copolymer of polylactic acid and polycaprolactone, for example, a multiblock copolymer represented by the following general formula (1) can be used. The polylactic acid may be any of poly-L-lactic acid, poly-D-lactic acid, and poly-DL-lactic acid. However, when considering application to medical use, in particular, poly-L-lactic acid. Is preferred.

  In the general formula (1), m is 5 to 100, preferably 10 to 50, more preferably an integer of 15 to 30, n is an integer of 5 to 100, preferably 10 to 50, more preferably 15 to 30; x is an integer of 2 to 500, preferably 5 to 200, more preferably 10 to 100.

For example, as shown in FIG. 1, the above multiblock copolymer is prepared by adding lactic acid (lactide) (LA) in the presence of a catalyst to the other end of polycaprolactone (PCL) having a protecting group (benzyl group or the like) at one end. It can be easily obtained by polymerizing to synthesize polylactic acid (PLA) -polycaprolactone diblock copolymer (oligomer), and after deprotection, self-polycondensation.
Note that the method described above is a simpler method with higher reproducibility than the method disclosed in Japanese Patent No. 3526856. The polylactic acid segment length (n) and the polycaprolactone segment length (m) can be easily controlled.

  In addition, the above-mentioned multiblock copolymer is an illustration to the last, and is not limited to the said form. For example, the terminal OH or H can be substituted with other functional groups, or other types of monomers may be polymerized so as to be connected to the above-described multiblock copolymer. That is, in the present invention, if the polylactic acid block and the polycaprolactone block have a multi-layered structure in part, void formation by alkaline hydrolysis as described below is possible.

(1.2. Formation of Multiblock Copolymer)
In the present invention, the multiblock copolymer obtained as described above is formed into a film by various methods. For example, after the multiblock copolymer is dissolved in a solvent such as chloroform, a thin film of the multiblock copolymer can be formed on the substrate by casting on a substrate such as glass and drying. it can. The thickness of the thin film obtained in this case is 5 μm to 100 μm, preferably 10 μm to 50 μm. Alternatively, in addition to the casting method described above, a thin film can be obtained by melting the multiblock copolymer and performing extrusion molding, press molding, or the like.

(1.3. Alkaline hydrolysis treatment of thin film)
In the present invention according to the first embodiment, the thin film obtained as described above is subjected to an alkali hydrolysis treatment to be hydrophilized. Here, the present inventors have found that a thin film into which water is immersed can be obtained depending on the conditions of the alkaline hydrolysis treatment. That is, voids can be generated in the thin film by the alkali hydrolysis treatment, and the voids communicate from the front surface to the back surface of the thin film over time. Thereby, it can hydrophilize from the surface of a thin film to the inside. Such a phenomenon is not confirmed in a thin film composed only of polylactic acid, and is a unique phenomenon observed when the above-described multi-block copolymer is used.

  As the alkali used for the alkali hydrolysis treatment, those having a pH of preferably 12 or more, more preferably 13 or more are used. If the pH is too low, the alkaline hydrolysis of the thin film does not proceed and the desired voids cannot be obtained. The type of alkali used is not particularly limited as long as the above pH can be realized, and sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like can be used.

  About the processing time of an alkali hydrolysis process, it can adjust with the thickness of the obtained thin film. For example, when a thin film having a thickness of about 20 μm is obtained, a hydrophilic porous membrane having voids communicating from the front surface to the back surface can be obtained by immersing in a 1 M aqueous sodium hydroxide solution for about 24 hours.

  Thus, a thin film is made into a hydrophilic porous membrane by alkali hydrolysis treatment. Whether or not the resulting hydrophilic porous membrane has voids communicating from the membrane surface to the membrane back surface can be judged by the above pH and alkali hydrolysis treatment time, and the cross-sectional state of the membrane is observed with an electron microscope or the like You can also judge directly. Alternatively, whether or not the voids communicate with each other can be easily determined by placing a water droplet on the surface of the membrane and observing whether or not the void penetrates.

  The “hydrophilic porous membrane” means a membrane through which water can permeate from the membrane surface to the membrane back surface or a membrane through which water can permeate. Even if it is a porous membrane, in the case of a lipophilic porous membrane, water stops on the membrane surface, and water cannot permeate and permeate from the membrane surface to the membrane back surface. In particular, by subjecting the polylactic acid-polycaprolactone multiblock copolymer to an alkali hydrolysis treatment, the surface thereof is hydrophilized, and the contact angle with water is 40 ° or less, preferably 30 ° or less, more preferably 25 ° or less. It becomes the film | membrane which water can osmose | permeate by having the surface which becomes, and also having a space | gap. For reference, on the surface of the polylactic acid-polycaprolactone multiblock copolymer that has not been hydrophilized, the contact angle with water is about 70 °.

(2. Second Embodiment)
The hydrophilic porous membrane of the present invention according to the second embodiment comprises a multi-block copolymer of polylactic acid and polycaprolactone, and has a void that communicates from the membrane surface to the membrane back surface. It is.

The first embodiment described above and the second embodiment are common in being a hydrophilic porous membrane, comprising a multi-block copolymer of polylactic acid and polycaprolactone, and having voids. ing. However, in the hydrophilic porous membrane according to the second embodiment, the void may be formed without depending on the alkali hydrolysis treatment. That is, after obtaining a thin film of a multi-block copolymer, voids are formed, and then the hydrophilic porous film according to the present invention is also obtained by subjecting the thin film having the voids to a hydrophilic treatment such as alkaline hydrolysis. A membrane can be obtained. Alternatively, a porous film having voids is obtained by forming a thin film while including a template for forming voids, and then removing the template, and thereafter, hydrophilicity such as alkali hydrolysis is applied to the porous membrane. The form which performs a conversion process may be sufficient.
However, as described in the first embodiment, according to the alkali hydrolysis treatment, since the hydrophilization treatment of the polylactic acid-polycaprolactone multiblock copolymer and the formation of voids can be performed simultaneously, it is convenient and preferable. .

  Thus, the hydrophilic porous membrane according to the present invention comprises a polylactic acid-polycaprolactone multiblock copolymer, and thus has excellent mechanical strength, flexibility, and biodegradability. Is excellent. Moreover, since it is a porous film | membrane hydrophilicized not only to the surface but the inside, its adhesiveness with respect to a wet surface is high. That is, it does not move in the living body, can be held at a predetermined position for a long time, and is appropriately decomposed in the living body.

<Medical anti-adhesion film>
As described above, the hydrophilic porous membrane according to the present invention does not move in the living body, can be held at a predetermined position for a long time, and is appropriately decomposed in the living body. Therefore, it can be suitably used as an anti-adhesion film for preventing adhesion of injured organs in vivo.

  When used as an anti-adhesion membrane, a laminate of a plurality of hydrophilic porous membranes may be used. That is, by forming a multilayer, the thickness of the hydrophilic porous membrane can be increased, and accordingly, the time until the membrane is completely biodegraded in a living body can be appropriately adjusted.

<Cell growth substrate>
The hydrophilic porous membrane according to the present invention can also be used as a base material for cell growth by utilizing its characteristics. That is, the hydrophilic porous membrane according to the present invention is hydrophilized to the inside, and cell reeds can extend not only to the membrane surface but also to the inside to allow cell proliferation to proceed. Further, as described above, since it is biodegradable, it can be easily used for a living body. Furthermore, since it has sufficient water permeability, it becomes possible to perform cell culture with multiple layers. For example, cells (artificial skin, etc.) cultured in a hydrophilic porous membrane can be transplanted directly into a living body.

<Method for producing hydrophilic porous membrane>
The hydrophilic porous membrane according to the present invention (particularly the hydrophilic porous membrane of the present invention according to the first embodiment) includes a step of obtaining a multi-block copolymer of polylactic acid and polycaprolactone, and the obtained multi It can be easily produced by a step of forming the block copolymer into a film and a step of forming voids in the film by subjecting the formed film to an alkali hydrolysis treatment. Details of each step are as described above, and a description thereof is omitted here.

  Hereinafter, although the hydrophilic porous membrane which concerns on this invention is further explained in full detail according to an Example, this invention is not limited to the specific form described in the following Examples.

<Application experiment to anti-adhesion membrane>
(1. Preparation of evaluation film)
(Example)
A polylactic acid-polycaprolactone multiblock copolymer was dissolved in chloroform at a concentration of 0.03 to 0.04 g / mL, and cast onto a glass plate. After casting, it was dried at room temperature under atmospheric pressure for 2 hours, and further dried at room temperature under vacuum for 12 hours. Then, it was immersed in water, the thin film was peeled off from the glass plate, and it dried at 50 degreeC under vacuum for 2 hours. The film thickness of the obtained thin film was 16 to 26 μm. The thin film was immersed in a 1M aqueous sodium hydroxide solution for 24 hours to prepare an evaluation film according to the example.

(Reference example)
An evaluation film according to a reference example was prepared in the same manner as in the example except that the thin film was immersed in a 1M sodium hydroxide aqueous solution for 12 hours.

(Comparative example)
An evaluation film according to a comparative example was prepared in the same manner as in the example except that polylactic acid was used without using the multiblock copolymer.

(2. Evaluation of membrane)
(2.1. SEM observation results)
FIG. 2A shows an SEM image showing the surface state of the evaluation film according to the example, and FIG. 2B shows an SEM image showing the surface state of the evaluation film according to the reference example. As is clear from FIG. 2, the evaluation film according to the example has a plurality of non-uniform voids having a diameter of about 10 μm formed on the surface. On the other hand, in the evaluation film according to the reference example, small holes were formed on the film surface. Note that voids / holes were not confirmed in the film according to the comparative example. That is, it has been found that the formation of voids by the alkali hydrolysis treatment is a phenomenon peculiar to the multiblock copolymer. Hereinafter, hydrophilicity evaluation etc. were performed about the evaluation film | membrane which concerns on an Example and a reference example.

(2.2. Evaluation of hydrophilicity)
For each of the evaluation film according to the example and the evaluation film according to the reference example, water was dropped to measure the contact angle and the hydrophilicity was evaluated. In the evaluation film according to the example, the dropped water was the film. It penetrated into the interior, and the contact angle could not be achieved. On the other hand, in the evaluation film | membrane which concerns on a reference example, the dripped water stayed on the surface and the contact angle was 25 degrees. For reference, when the contact angle with respect to the thin film surface of the polylactic acid-polycaprolactone multiblock copolymer not subjected to alkali hydrolysis was measured, it was 70 °.

(2.3. Performance evaluation as anti-adhesion membrane)
The evaluation membrane according to the example and the evaluation membrane according to the reference example were cut to 20 × 20 mm, gas sterilized, and placed so as to be in close contact with the surface of the affected liver in the abdominal cavity of the rat. In this experiment, the evaluation film according to the example and the evaluation film according to the reference example were installed at different locations on the liver of one rat. After a certain period, the abdomen of the rat was restarted and the state of adhesion was observed. Thereafter, the liver was removed and the tissue state of the liver surface was observed using a microscope. The results are shown in Table 1 below and FIG.

As shown in Table 1, when the evaluation membrane according to the reference example is used as an adhesion prevention membrane, it is difficult to stop at a predetermined position in the living body. Was out of position. Some were misaligned until completely removed from the affected area, and strong adhesion was confirmed. Moreover, almost no decomposition of the evaluation film could be confirmed. On the other hand, when the evaluation film | membrane which concerns on an Example was used as an adhesion prevention film | membrane, it was closely_contact | adhering without shifting | deviating from the original position even after 6 days. In addition, although adhesion was seen about one part, it was the very small thing of the grade which peels when the said adhesion part is pulled lightly. In the evaluation film according to the example, the decomposition of the film could be confirmed, and the film only remained thin.
Furthermore, after 2 weeks, the stomach was resumed and the state was observed. As a result, in the evaluation membrane according to the reference example, almost no degradation of the membrane could be confirmed as in 6 days. On the other hand, in the evaluation film according to the example, the film was completely decomposed.

  FIG. 3 is an observation diagram showing the state of the liver surface 6 days after the evaluation membrane according to the example is installed. As can be seen from FIG. 3, the membrane was decomposed while being in close contact with the liver surface, and the nucleus of the cell had entered the membrane.

  As described above, it was proved that the evaluation film according to the example was appropriately biodegradable and excellent in adhesion to the wet surface and could be suitably used as an adhesion preventing film.

<Application experiment as a cell growth substrate>
The polylactic acid-polycaprolactone multi-block copolymer thin film was converted into a hydrophilic porous membrane by alkaline hydrolysis treatment, and cells (HeLa cells) were cultured on the obtained hydrophilic porous membrane. FIG. 4A shows the state of the cells in the membrane after cell culture, and FIG. 4B shows a composite photograph of the cells and the membrane. As is clear from FIG. 4, the cells have grown into the inside through the voids of the membrane. That is, it was found that the hydrophilic porous membrane according to the present invention can also be applied as a cell growth substrate.

<Relationship between alkali pH and void formation>
A polylactic acid-polycaprolactone multi-block copolymer thin film having a thickness of 30 μm was obtained. The obtained thin film was immersed in a sodium hydroxide aqueous solution having a pH of 11 to 13, and the critical point of pH was specified as to whether or not the alkaline hydrolysis proceeds. Experiments have shown that alkaline hydrolysis proceeds appropriately, and the pH at which the voids can be appropriately formed in the thin film is 12 or more.

  Although the present invention has been described with reference to the most practical and preferred embodiments at the present time, the invention is not limited to the embodiments disclosed herein. The hydrophilic porous membrane and its manufacturing method, medical adhesion preventing membrane, which can be appropriately changed without departing from the gist or concept of the invention that can be read from the claims and the entire specification, Cell growth substrates are also to be understood as being included within the scope of the present invention.

  The hydrophilic porous membrane according to the present invention has biodegradability and excellent adhesion to a wet surface. For example, it can be held in a predetermined position for a long period of time without being displaced in the living body, and is appropriately decomposed in the living body. Because of these characteristics, it can be widely used in the medical field, such as an anti-adhesion membrane and a cell growth substrate.

Claims (3)

Obtaining a multi-block copolymer of polylactic acid and polycaprolactone;
Forming the obtained multiblock copolymer into a film;
A step of generating voids in the membrane by subjecting the formed membrane to an alkali hydrolysis treatment with an alkali having a pH of 12 or more ;
Equipped with a,
The alkali hydrolysis treatment is performed until the gap communicates from the front surface to the back surface of the membrane.
A method for producing a hydrophilic porous membrane. The method for producing a hydrophilic porous membrane according to claim 1, wherein the thickness of the membrane is 5 μm or more and 100 μm or less. In the step of obtaining the multiblock copolymer, a diblock copolymer of the polylactic acid and the polycaprolactone is synthesized, and the diblock copolymer is subjected to post-polymerization to obtain the multiblock copolymer. A method for producing a hydrophilic porous membrane according to claim 1 or 2 .