JP2011115097A - Device for evaluating cell permeability of polymer compound or complex thereof, and method for evaluating the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for evaluating the cell permeability of a polymer compound or its complex simply and more accurately, and a method for evaluating the cell permeability of the polymer compound or its complex simply and more accurately. <P>SOLUTION: This device for evaluating the cell permeability of the polymer compound or its complex is provided by having a first region part for putting-in the polymer compound or its complex, a second region part adjacent to the first region part, for housing the polymer compound or its complex permeated from the first region and a permeating substrate material arranged on the boundary of the first and second region parts. The permeating substrate material is constituted by a fiber prepared by an electric field spinning method by using gelatin, and also constituted as capable of forming the single layer of cells closely bonded with the one side surface of the first region part side and capable of permeating the polymer compound or its complex. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a device for evaluating cell permeability of a polymer compound or a complex thereof and a method for evaluating cell permeability thereof, in particular, a fiber made by an electrospinning method using gelatin and the first region side. Cell permeation evaluation apparatus having a permeable substrate capable of forming a single-layer cell tightly bonded to one side of the substrate and configured to be permeable to a polymer compound or a complex thereof, and this permeable substrate The present invention relates to a cell permeability evaluation method.

  Development of vectors and carriers for reliably delivering substances such as proteins, drugs or genes to target sites of living organisms has been actively conducted. For example, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses have been developed as vectors for introducing a target gene into target cells. However, since viral vectors have problems such as difficulty in mass production, antigenicity, and toxicity, liposome vectors and peptide carriers with few such problems are attracting attention. Liposome vectors also have the advantage that directivity with respect to target sites can be improved by introducing functional molecules such as antibodies, proteins, and sugar chains on the surface.

  On the other hand, when a high molecular compound such as a liposomal vector or a liposomal vector encapsulating a substance or a complex thereof is administered into blood, reaching the target site from the blood becomes a major issue. Vascular endothelial cells form tight junctions between cells in many tissues, and the gap between vascular endothelial cells is extremely narrow, about 0.4 nm to about 4 nm. Therefore, a polymer compound or a composite thereof administered into blood is used. It is difficult for the body to reach the target site through this gap. Therefore, research and development have been conducted on a technique for allowing a polymer compound or a complex thereof to permeate into a cell and reach a target site by utilizing functions such as transcytosis of a cell.

  In such research and development, a technique for evaluating the cell permeability of a substance, particularly, the cell permeability of a polymer compound or a complex thereof is important. As a conventional method for evaluating the cell permeability of a polymer compound, Millicell (Millipore), a cell culture insert or a multiwell insert (all of which are BD Biosciences), which is a conventionally used cell permeability evaluation apparatus, or a transformer A method using a well (Corning) or the like is performed. All of these conventionally used cell permeability evaluation apparatuses have the same structure, and as shown in FIG. 2, the conventional cell permeability evaluation apparatus 5 includes polyesters such as polyethylene terephthalate (PET), It comprises an insert 9 having a membrane 8 made of polycarbonate or the like at its base, an upper layer region 6 and a lower layer region 7 that are vertically partitioned by the insert 9, and a well plate 10 that surrounds the lower layer region 7. The membrane 8 has a diameter of 0.4 μm. 1.0 μm, 3.0 μm, or 8.0 μm. In the case of evaluating the cell permeability of a polymer compound or the like using the conventional cell permeability evaluation apparatus 5, after the cells are monolayer cultured on the upper layer region 6 side using the membrane 8 as a base material, Contact the sample. When the sample has cell permeability, the sample passes through the cells and the membrane 8 and moves to the lower layer region 7, and therefore the cell permeability of the sample can be evaluated by detecting the sample in the lower layer region 7. .

  Furthermore, in addition to the above configuration, an apparatus (Patent Document 1) that includes a sensor that detects a substance that has migrated to the lower layer area 7, and an apparatus that has an adsorbent that adsorbs the substance that has migrated from the upper layer area 6 to the lower layer area 7. (Patent Document 2) has been developed.

JP-T-2006-505278 JP 2009-5608 A

  However, in the membrane 8 provided in the conventional cell permeability evaluation apparatus 5, when the diameter (pore diameter) of the minute through hole of the membrane 8 is 1.0 μm or less, the high molecular compound such as liposome or the complex thereof is the membrane 8. Can hardly penetrate. In addition, when the pore diameter of the membrane 8 is 3.0 μm or more, the cultured cells cannot form a single layer on the upper layer region 6 side of the membrane 8, and further, within the pores of the membrane 8 and the lower layer region 7 of the membrane 8. Therefore, it is difficult to accurately evaluate the cell permeability of a polymer compound or a complex thereof using the conventional cell permeability evaluation apparatus 5.

  The present invention has been made to solve such problems, and is a device for simply and more accurately evaluating cell permeability of a polymer compound or a complex thereof, and a polymer compound or a complex thereof. An object of the present invention is to provide a simple and more accurate method for evaluating cell permeability.

  As a result of diligent research, the present inventors have found that a permeable substrate composed of fibers produced by an electrospinning method using gelatin transmits a polymer compound or a complex thereof, The inventors found that monolayer cells tightly bonded to one surface can be formed, and completed the following inventions.

(1) An apparatus for evaluating the cell permeability of a polymer compound or a complex thereof, wherein the first region portion into which the polymer compound or the complex is introduced is adjacent to the first region portion and the first region portion. A second region containing a polymer compound or a composite thereof permeated from one region, and a permeable substrate disposed on a boundary between the first region and the second region; The permeable substrate is formed of fibers produced by electrospinning using gelatin, and can form single-layer cells tightly bonded to one side of the first region portion side. The apparatus, which is configured to be permeable to a polymer compound or a complex thereof.

(2) The apparatus according to (1), wherein the diameter D of the fiber produced by electrospinning using gelatin is 500 nm ≦ D ≦ 1000 nm, and the distance I between the fibers is 5 μm ≦ I ≦ 20 μm.

(3) The device according to (1) or (2), wherein the polymer compound or a complex thereof is a lipid or a lipid membrane structure.

(4) A method for evaluating the cell permeability of a polymer compound or a complex thereof, which is composed of fibers made by an electrospinning method using gelatin and can penetrate a polymer compound or a complex thereof. Forming a monolayer cell tightly bonded to one side of a permeable substrate, a step of contacting a polymer compound or a complex thereof with the monolayer cell, and a polymer compound with the monolayer cell Alternatively, the method comprises a step of evaluating cell permeability from a polymer compound or a complex thereof obtained by contacting the complex and passing through the monolayer cell and the permeable substrate.

(5) A method for evaluating the cell permeability of a polymer compound or a composite thereof, which is composed of fibers made by electrospinning using gelatin and can penetrate a polymer compound or a composite thereof. A step of obtaining a polymer compound or a composite thereof permeating through the transparent substrate by contacting a polymer compound or a composite thereof on one side of the transparent substrate configured in the above; A step of forming a monolayer cell tightly bonded to one side, and a polymer compound or a complex thereof permeated through the monolayer cell and the permeable substrate by contacting the monolayer cell with a polymer compound or a complex thereof. A polymer compound or a composite thereof that has passed through the obtained permeable substrate, and a polymer compound or a composite thereof that has passed through the obtained single-layer cell and the permeable substrate. And a step of evaluating the cell permeability by comparing the door, said method.

(6) The diameter D of the fiber produced by electrospinning using gelatin is 500 nm ≦ D ≦ 1000 nm, and the distance I between the fibers is 5 μm ≦ I ≦ 20 μm, as described in (4) or (5) the method of.

(7) The method according to any one of (4) to (6), wherein the polymer compound or a complex thereof is a lipid or a lipid membrane structure.

  According to the present invention, the cell permeability of a polymer compound or a complex thereof that could not be evaluated with a conventional cell permeability evaluation apparatus can be easily evaluated.

It is a conceptual diagram which shows the basic composition of the cell permeability evaluation apparatus 1 of the high molecular compound or its composite_body | complex in this embodiment. It is a conceptual diagram which shows the aspect of the conventional cell permeability evaluation apparatus 5 conventionally used when evaluating the cell permeability of a high molecular compound. It is the figure (photograph) which observed the gelatin nanofiber sheet with the electron microscope. It is a figure which shows the cell-free liposome permeability | transmittance in the PET membrane 8 of the permeable base material 4 of the cell permeability evaluation apparatus 1 and the conventional cell permeability evaluation apparatus 5 which concern on this embodiment. It is sectional drawing (photograph) of the MBEC4 cell layer cultured in the permeable base material 4 of the cell-permeability evaluation apparatus 1 which concerns on this embodiment. It is the figure (photograph) which detected the ZO-1 protein and nucleus of the MBEC4 cell cultured in the permeable base material 4 of the cell permeability evaluation apparatus 1 which concerns on this embodiment. It is a mimetic diagram showing one mode of a method of performing TER measurement in cell permeability evaluation device 1 concerning this embodiment. MBEC4 cells cultured on the permeable substrate 4 of the cell permeability evaluation apparatus 1 according to the present embodiment, MBEC4 cells cultured on the PET membrane 8 of the conventional cell permeability evaluation apparatus 5, and the conventional cell permeability evaluation apparatus 5 is a diagram showing a TER measurement result of MFLN4 cells cultured on a membrane 8 made of PET. It is a figure which shows the cell-free liposome permeability | transmittance in the membrane 8 made from PET of the conventional cell permeability evaluation apparatus 5 whose pore diameter of a membrane is 0.4 micrometer, 1.0 micrometer, 3.0 micrometer, and 8.0 micrometer, respectively. In the figure, the vertical axis represents the permeability (%) of cell-free liposomes, and the horizontal axis represents the transition time (hours). The ZO-1 protein of MBEC4 cells cultured on the PET membrane 8 (pore size 0.4 μm) of the conventional cell permeability evaluation apparatus 5 is observed by 2.0 μm at an observation depth from the upper part of the membrane 8 to the lower part of the membrane 8. It is the figure (photograph) observed by changing. The MBEC4 cell ZO-1 protein cultured in the PET membrane 8 (pore size: 1.0 μm) of the conventional cell permeability evaluation apparatus 5 is observed at a depth of 2.0 μm from the upper part of the membrane 8 to the lower part of the membrane 8. It is the figure (photograph) observed by changing. The MBEC4 cell ZO-1 protein cultured on the PET membrane 8 (pore size 8.0 μm) of the conventional cell permeability evaluation apparatus 5 has an observation depth of 2.0 μm from the upper part of the membrane 8 to the lower part of the membrane 8. It is the figure (photograph) observed by changing. The ZO-1 protein and nuclei of MBEC4 cells cultured on the PET membrane 8 (pore size 8.0 μm) of the conventional cell permeability evaluation apparatus 5 have an observation depth of 1.0 μm from the upper part of the membrane 8 to the lower part of the membrane 8. It is the figure (photograph) observed by changing each one. The ZO-1 protein of MBEC4 cells cultured in gelatin-coated PET membrane 8 and collagen-coated PET membrane 8 (both membrane pore diameters are 3.0 μm) were placed on top of membrane 8, inside membrane 8, It is the figure (photograph) observed in the lower part of the membrane 8. FIG.

  Hereinafter, a cell permeability evaluation apparatus and a cell permeability evaluation method for a polymer compound or a complex thereof according to the present invention will be described in detail.

  First, an embodiment of a cell permeability evaluation apparatus for a polymer compound or a complex thereof according to the present invention will be described with reference to the drawings. FIG. 1 is a conceptual diagram illustrating a basic configuration of a cell permeability evaluation apparatus 1 in the present embodiment. As shown in FIG. 1, the cell permeability evaluation apparatus 1 mainly includes a first region portion 2 into which a polymer compound or a complex thereof is charged, and is adjacent to the first region portion 2, and from the first region portion 2. It is comprised from the 2nd area | region part 3 which accommodates the polymer compound or its composite_body | complex to permeate | transmit, and the permeable base material 4 arrange | positioned on the boundary of the 1st area | region part 2 and the 2nd area | region part 3. . That is, the first region portion 2 and the second region portion 3 are adjacent to each other.

  The first region portion 2 is a region into which a polymer compound or a complex thereof is introduced, and may have a configuration for holding the introduced polymer compound or the complex for a certain period of time. As such a configuration, for example, an aspect such as a container capable of holding a liquid can be cited. The material constituting the container is not particularly limited, and a material excellent in water resistance, corrosion resistance, chemical resistance, and the like is preferable. Examples of such a material include glass, plastic, and stainless steel.

  Examples of the polymer compound in the present invention include lipids, proteins, peptides, nucleic acids, polysaccharides, plastics, silicon, synthetic rubbers and the like, and lipids are preferable. In addition, examples of the complex of the polymer compound include a lipid membrane structure, a lipid membrane structure containing a low molecular compound or a polymer compound, a dimeric protein in which two molecules of proteins are bound by a hydrophobic domain, and the like. Can be mentioned. Examples of lipid membrane structures include liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, string micelles, and irregular layered structures. Examples of liposomes include Single membrane liposomes such as SUV (small unilamellar vesicle), LUV (large unilamellar vesicle), GUV (giant unilamellar vesicle), multilamellar liposomes (MLV), or those having functional molecules introduced therein, and further drugs , Proteins, genes and the like.

  As a method of introducing the polymer compound or the complex thereof into the first region portion 2, a method that can be appropriately selected by those skilled in the art can be used. Examples of such a method include a method in which a polymer compound or a complex thereof is dissolved or suspended in a buffer solution such as a HEPES buffer, and this suspension is injected using a pipette or the like. .

  The second region portion 3 is charged into the first region portion 2 and accommodates the formed single-layer cells and the permeable base material 4 described later, or a polymer compound or a complex thereof that has permeated the permeable base material 4. It is only necessary to have a structure that holds the permeated polymer compound or a complex thereof. As such a configuration, the same aspect as that of the first region portion 2 described above can be exemplified.

  The permeable substrate 4 is composed of fibers produced by an electrospinning method using gelatin. The electrospinning method is one of the methods for producing ultrafine fibers (nanofibers). A syringe needle having an injection syringe filled with a polymer solution as a raw material and an electrode for collecting the fibers are arranged facing each other and injected. After filling the syringe with the polymer solution, the polymer solution filled with the syringe pump is pushed out from the syringe needle while applying a high voltage between the syringe needle and the electrode. This is a method of accumulating on the fiber electrode protruding in a spiral shape.

  In the present invention, fibers can be produced by electrospinning using gelatin by combining instruments and methods that can be appropriately selected by those skilled in the art, such as injection syringes, syringe pumps, syringe needles, high voltage generators, and the like. These may be combined to produce an electrospinning apparatus, which may be used, or may be performed using a commercially available electrospinning apparatus. Also, the origin of the raw material gelatin, the type of solvent used to prepare the gelatin solution, the concentration of the gelatin solution, the inner diameter of the syringe needle, the speed at which the gelatin solution is extruded, the type and shape of the electrode for collecting the fiber, the tip of the syringe needle and the fiber The distance to the electrode to be collected, the magnitude of the voltage to be applied, etc. are appropriately determined according to the instrument and method used for producing the fiber by the electrospinning method, the required amount of fiber, the type of the polymer compound to be evaluated or its complex, etc. Can be set.

  The diameter of the fiber produced by electrospinning using gelatin and the distance between the fibers can be appropriately measured according to a conventional method, and can be measured using, for example, an electron microscope. In the present invention, the value of the diameter D and the distance I between the fibers produced by the electrospinning method using gelatin are not particularly limited, but may be 500 nm ≦ D ≦ 1000 nm, or 5 μm ≦ I ≦ 20 μm. preferable. Further, the value of the thickness T of the transmissive substrate is not particularly limited, but is preferably 150 μm ≦ T ≦ 250 μm.

  Although the permeable base material 4 is arrange | positioned on the boundary of the 1st area | region part 2 and the 2nd area | region part 3, the aspect which partitions off the 1st area | region part 2 and the 2nd area | region part 3 may be sufficient as the aspect. The aspect which becomes a part on the boundary of the 1st field part 2 and the 2nd field part 3 may be sufficient. As an aspect in which the permeable substrate 4 becomes a part on the boundary between the first region portion 2 and the second region portion 3, for example, a polymer compound such as glass, plastic, stainless steel, or a composite thereof is used. A mode in which the first region portion 2 and the second region portion 3 are partitioned by a material that does not allow permeation and a part thereof is replaced with the permeable base material 4 can be exemplified.

  The permeable substrate 4 is configured to be permeable to a polymer compound or a complex thereof. In the present invention, the term “configured to be permeable to a polymer compound or a complex thereof” refers to a polymer. It means that part or all of a compound or a complex thereof is configured to permeate.

  The permeable substrate 4 is further configured to be capable of forming a single-layer cell that is tightly bonded to one surface on the first region 2 side. Here, the “monolayer cell” refers to a cell group forming one layer by arranging cells without overlapping each other. In addition, “tight junction” means that tight junction-binding proteins such as occludin and claudin are densely arranged in the gap between adjacent cells, thereby preventing the passage of substances. It is a cell-to-cell binding mode.

  As a method for confirming whether or not a monolayer cell has been formed, for example, after staining the nuclei of cultured cells with Hoechst 33342, images are obtained by changing the observation depth using a fluorescence microscope. In the cross-sectional view of the originally constructed cell layer, in addition to the method for confirming whether the cell layer forms a single layer, ZO-1 protein, which is a cell membrane protein of cultured cells, is immunofluorescently stained, A method to check whether fluorescence is observed at the observation depth corresponding to the inside or the bottom (back side) of a culture substrate such as a permeable substrate or membrane by observing with a focus laser microscope. Can be mentioned.

  Examples of the method for confirming whether tight junctions have been formed include, for example, a method for confirming using a fluorescence microscope after immunofluorescent staining of ZO-1 protein, which is a tight junction marker protein. A method of confirming the cultured cells by measuring transmembrane electrical resistance (TER) can be mentioned.

  The TER measurement is a method for confirming the tight bond formation state in the cell layer by measuring the electrical resistance between the electrodes arranged with the cell layer interposed therebetween. This measurement method is based on the fact that when a tight bond is formed between cells, the electrical resistance increases due to the non-conductivity of the cell membrane. When performing TER measurement in the present invention, as shown in FIG. 7, the upper layer side region (in FIG. 1, the first region portion 2 corresponds to the upper layer region 6 in FIG. 2) and the lower layer side region (in FIG. In FIG. 1, the second region 3 corresponds to the lower region 7 in FIG. 2), and the electrical resistance may be measured.

  In the present invention, the method for forming a monolayer cell tightly bonded to one side of a permeable substrate can be appropriately performed according to a conventional method. Examples of such a method include a method of incubating under appropriate culture conditions in a state where the cell suspension is in contact with one surface of the permeable substrate. Cell culture conditions for forming monolayer cells can be appropriately set according to the type of cells used, the type of culture solution, the initial cell concentration, the area of the permeable substrate, and the like. Moreover, the kind of cell to be used is not particularly limited as long as it can be tightly bound, and examples thereof include epithelial cells such as vascular endothelial cells and skin epithelial cells.

  When single-layer cells tightly bonded to the first region portion 2 side of the permeable base material 4 are formed, the polymer compound or the complex introduced into the first region portion 2 After permeating the monolayer cells formed on one side, the permeation base 4 is permeated and accommodated in the second region 3.

  In addition, when calculating | requiring the transmittance | permeability of the high molecular compound or its composite_body | complex in the permeable base material 4, after putting a high molecular compound or its composite_body | complex in the 1st area | region part 2 and leaving it for a fixed time, it is 2nd area | region, for example. It can be determined by measuring the amount of the polymer compound or complex thereof present in part 3 and calculating the percentage of the measured amount with respect to the amount added.

Next, the cell permeability evaluation method for a polymer compound or a complex thereof according to the present invention is a method for evaluating the cell permeability of a polymer compound or a complex thereof,
(I) Single-layer cells that are tightly bonded to one side of a permeable substrate that is composed of fibers made by electrospinning using gelatin and is permeable to a polymer compound or a complex thereof. Step of forming (monolayer cell forming step)
(Ii) A step of bringing the polymer compound or a complex thereof into contact with the monolayer cell (sample contact step)
(Iii) A step of evaluating cell permeability from a polymer compound or a complex thereof obtained by contacting the monolayer cell with a polymer compound or a complex thereof and permeating the monolayer cell and the permeable substrate (cells) Permeability evaluation process)
The above steps (i) to (iii) are included.

  (Ii) In the sample contacting step, the method of bringing a polymer compound or a complex thereof into contact with a monolayer cell cultured on a permeable substrate can be appropriately performed according to a conventional method. Examples of such a method include a method of adding a high molecular compound or a complex thereof dissolved or suspended in a HEPES buffer solution using a pipette after exchanging the cell culture solution with a Krebs buffer solution. Can be mentioned.

  (Iii) As a method for evaluating cell permeability in the cell permeability evaluation step, for example, (ii) the amount of the polymer compound or its complex brought into contact with the monolayer cell in the sample contact step, A method for comparing the amount of the polymer compound or the complex thereof permeated through the layer cell and the permeable substrate can be mentioned.

Next, one of the different aspects of the cell permeability evaluation method of the polymer compound or the complex thereof according to the present invention is a method for evaluating the cell permeability of the polymer compound or the complex thereof,
(Iv) A polymer compound or composite thereof on one side of a permeable substrate made of fibers produced by electrospinning using gelatin and configured to be permeable to the polymer compound or composite thereof For obtaining a polymer compound or a complex thereof that has passed through the permeable substrate by contacting the substrate (first permeation sample obtaining step)
(V) forming a monolayer cell tightly bonded to one side of the permeable substrate (monolayer cell formation step)
(Vi) A step of obtaining a polymer compound or a complex thereof permeated through the monolayer cell and the permeable substrate by contacting the monolayer cell with a polymer compound or a complex thereof (second permeation sample obtaining step) )
(Vii) Cells obtained by comparing the obtained polymer compound or a complex thereof permeated through the permeable substrate with the obtained polymer compound or a complex thereof permeated through the obtained monolayer cell and the permeable substrate. Process for evaluating permeability (cell permeability evaluation process)
The above steps (iv) to (vii) are included.

  (Vii) As a method for evaluating cell permeability in the cell permeability evaluation step, for example, (iv) measuring the amount of the polymer compound or complex thereof acquired in the first permeation sample acquisition step, vi) After measuring the amount of the polymer compound or complex thereof obtained in the second permeation sample obtaining step, a method of comparing these measured amounts can be mentioned. In this case, (iv) the ratio of the amount of the polymer compound or the complex acquired in the second transmission sample acquisition step to the amount of the polymer compound or the complex acquired in the first transmission sample acquisition step is large. It can be evaluated that the higher the cell permeability of the polymer compound or the complex as a sample is, the greater the value is.

  The amount of the polymer compound or the complex thereof can be appropriately measured according to a method that can be selected by those skilled in the art. As such a method, for example, a high molecular compound or a complex thereof is previously labeled with a radioisotope (RI), a fluorescent dye, or a peptide recognized by a detectable antibody, and a liquid scintillation counter, The label can be detected and measured using a microscope, a fluorescence microscope, an immunostaining method, or the like.

  Hereinafter, an apparatus for evaluating cell permeability of a polymer compound or a complex thereof and a method for evaluating cell permeability of a polymer compound or a complex thereof according to the present invention will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Production and preparation of experimental materials (1) Production of permeable substrate Gelatin (Nitta Gelatin Co., Ltd .; derived from cattle bone; molecular weight 100000; isoelectric point 5) was converted to hexafluoroisopropanol (Wako Pure Chemical Industries, Ltd.) A gelatin solution was prepared by dissolving to 7% (w / w), and filled in a 5 mL injection syringe of the electrospinning apparatus produced by the present inventors. A syringe needle having an inner diameter of 0.51 mm was connected to the injection syringe, and a 5 cm × 5 cm square metal plate covered with aluminum foil was installed as an electrode for collecting fibers. The tip of the syringe needle, which is an electrode, and a metal plate are placed 22 cm apart, and a gelatin solution filled in an injection syringe is extruded at a rate of 9 mL / hour while applying a voltage of 14 kV between these electrodes, and the aluminum foil of the metal plate An accumulation of gelatin fibers was obtained on top. The obtained gelatin fiber aggregate was subjected to heat treatment using a vacuum dryer (vacuum dryer DN-30S; Sato Vacuum Co., Ltd.) at 160 ° C. for 24 hours to thermally dehydrate and crosslink the gelatin nanofiber sheet. Obtained.

  FIG. 3 shows the result of observation of the gelatin nanofiber sheet using an electron microscope after the heat treatment. As shown in FIG. 3, it was confirmed that the gelatin nanofiber sheet formed a sheet in which gelatin fibers were entangled randomly.

  Further, when the diameter of the fiber of the gelatin nanofiber sheet, the distance between the fibers and the thickness thereof were measured using an electron microscope, the diameter D of the fiber was 500 nm ≦ D ≦ 1000 nm, and the distance I between the fibers was 5 μm ≦ I ≦. Since 20 μm and the thickness T of the gelatin nanofiber sheet was 150 μm ≦ T ≦ 250 μm, it was confirmed that the gelatin nanofiber sheet was permeable.

(2) Production of cell permeability evaluation apparatus Conventional cell permeability evaluation apparatus 5 {Millicell; Millipore, membrane material: made of polyethylene terephthalate (PET), membrane pore size: 3.0 µm, 24 well} The PET membrane was excised using a cutter knife. Subsequently, the gelatin nanofiber sheet produced in this Example (1) was adhered to the site where the PET membrane was excised using Aron Alpha for Plastic (Toagosei Co., Ltd.), so that gelatin nanofibers were used instead of the PET membrane. An insert having a permeable substrate 4 composed of a fiber sheet was produced. By installing this insert in a 24-well plate, a cell permeability evaluation apparatus 1 having a permeable substrate 4 made of a gelatin nanofiber sheet instead of a PET membrane was produced (FIG. 1).

(3) Preparation of Krebs Buffer A Krebs buffer (pH 7.3) having the following composition was prepared using sterile water as a solvent.

Composition of Krebs buffer KCl (Wako Pure Chemical Industries) 4.8 mmol / L
KH ₂ PO ₄ (Wako Pure Chemical Industries, Ltd.) 1.0 mmol / L
MgSO ₄ (Wako Pure Chemical Industries, Ltd.) 1.2 mmol / L
2- [4- (2-Hydroxyethyl) -1-piperazinyl] etheresufonic acid (HEPES) (Wako Pure Chemical Industries, Ltd.)
12.5mmol / L
CaCl ₂ (Wako Pure Chemical Industries) 1.5mmol / L
NaCl (Nacalai Tesque) 120.0mmol / L
NaHCO ₃ (Wako Pure Chemical Industries, Ltd.) 23.8 mmol / L
Glucose (Wako Pure Chemical Industries) 5.0mmol / L

(4) Preparation of RI-labeled liposomes Egg yolk phosphatidylcholine {L-α-Phosphatidylcholine, Egg (Powder); Avanti Polar Lipids} and cholesterol {Cholesterol (Powder); Avanti Polar Lipids} and egg yolk phosphati : Cholesterol = 7: 3 was mixed to prepare a mixed lipid powder. Subsequently, the prepared mixed lipid powder was dissolved in ethanol so as to be 1 nmol / μL, and 137.5 μL of this was put into a glass test tube, and ³ H-cholesteryl ether (CHE), which is a radioisotope (RI), was added. ) After adding 2.5 million dpm to 5 million dpm and 112.5 μL of chloroform and vortexing for a short time, the ethanol solvent was removed using a nitrogen gas stream according to a conventional method. Subsequently, 250 mL of ethanol was added to dissolve the lipid, and the ethanol solvent was removed again using a nitrogen gas stream in accordance with a conventional method to prepare a lipid membrane labeled with RI. 250 μL of 10 mmol / L HEPES buffer solution (pH 7.4) prepared according to a conventional method using HEPES (Wako Pure Chemical Industries, Ltd.) was added to the lipid membrane and left to hydrate for 15 minutes at room temperature. RI labeled liposomes were prepared by sonicating for 30 seconds.

(5) Confirmation of the size of RI-labeled liposomes RI-unlabeled liposomes were prepared by the method described in this Example (4) without adding RI, and a dynamic light scattering measurement device (Zetasizer Nano ZS; Malvern) As a result of measuring the size, the diameter of the liposome was about 100 nm. This revealed that the size of the RI-labeled liposome prepared in this Example (4) was about 100 nm in diameter.

<Example 2> Confirmation of cell-free liposome permeability in the permeable substrate 4 of the cell permeability evaluation apparatus 1 The cell permeability evaluation apparatus 1 prepared in Example 1 (2) and three types of conventional membrane pore diameters are different. In Example 1 (3), the cell permeability evaluation apparatus 5 (Millicell; Millipore Corporation, membrane material: PET, 24 wells, membrane pore diameter: 0.4 μm, 3.0 μm, 8.0 μm) The prepared Krebs buffer was added at 100 μL / well, and the RI-labeled liposome prepared in Example 1 (4) was added at 100 μL (about 1 to 2 million dpm) / well. Two hours later, the Krebs buffer was recovered from the lower layer side, and the amount of RI contained in the recovered Krebs buffer was measured using a liquid scintillation counter TRI-CARB 1600TR (PACKARD). By calculating the ratio of the measured RI amount to the RI amount of the liposome added to the upper layer side as a percentage, the permeability substrate 4 of the cell permeability evaluation apparatus 1 or the PET of the conventional cell permeability evaluation apparatus 5 is used. It was set as the liposome permeability (cell-free liposome permeability) when the cells were not cultured in the manufactured membrane. The result is shown in FIG.

  As shown in FIG. 4, the cell-free liposome permeability was about 17.5% in the cell permeability evaluation apparatus 1, whereas that in the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 0.4 μm. It was about 12% in the conventional cell permeability evaluation apparatus 5 having 0.17% and a membrane pore diameter of 3.0 μm, and about 19% in the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 8.0 μm.

  From the above results, the permeable substrate 4 of the cell permeability evaluation apparatus 1 in which no cultured cells are present is comparable to a PET membrane having a membrane pore diameter of 8.0 μm in the conventional cell permeability evaluation apparatus 5 in which no cultured cells are present. It was confirmed that about 17.5% of the liposomes permeate the permeable substrate 4.

<Example 3> Confirmation of the state of cells cultured in the permeable substrate 4 of the cell permeability evaluation apparatus 1 (1) The permeable substrate 4 of the cell permeability evaluation apparatus 1 and the PET of the conventional cell permeability evaluation apparatus 5 Cell culture on membrane membrane MBEC4 cells derived from mouse brain capillary endothelial cells and progenitor cells of vascular endothelial cells provided by Takashi Tsuruo and Mikihiko Naito of the Institute for Molecular Cell Biology, The University of Tokyo Mouse Fetal Lung-Derived Vascular Endothelial Cells {Mouse Fetal Lung Mesenchyme-4 (MFNL4) Cells; Seven Hills Bioagents}} in Dulbecco's Modified Eagle's Medium (DMEM Medium, 300 mL, respectively) It was suspended to become. The suspension of the MBEC4 cells is divided into an upper region of the cell permeability evaluation device 1 and the conventional cell permeability evaluation device 5 (Millicell; Millipore, membrane material: PET, membrane pore size: 0.4 μm, 24 wells). The solution was added at 400 μL / well. The suspension of MFLN4 cells is 400 μL / well in the upper layer side of the conventional cell permeability evaluation apparatus 5 (Millicell; Millipore, membrane material: PET, membrane pore size: 0.4 μm, 24 wells). I put it to be. These were statically cultured on a gelatin nanofiber sheet or a PET membrane at 37 ° C. for 3 to 5 days in a 5% CO ₂ atmosphere.

(2) Confirmation of location of MBEC4 cells in permeable substrate 4 of cell permeability evaluation apparatus 1 Hoechst33342 (DAIJINDO), which is a fluorescent dye for staining nuclei, is 1 μg / mL in this example (1). ) Was added to the region on the upper layer side of the cell permeability evaluation apparatus 1 in which MBEC4 cells were cultured, and cultured for 15 minutes under the culture conditions described in this Example (1). Subsequently, using an EM-CCD camera (Hamamatsu Photonics) and a fluorescence microscope (TE2000; Nikon), the fluorescence of Hoechst 33342 was detected by changing the observation depth by 15 μm, and an image was acquired. Then, based on the acquired image, sectional drawing of the cell layer in the permeable base material 4 was constructed | assembled. The result is shown in FIG.

  As shown in FIG. 5, in the MBEC4 cell layer in the permeable base material 4, it became clear that the nucleus shown by the fluorescence of Hoechst33342 is almost one layer. From this result, it was confirmed that when MBEC4 cells are cultured in the permeable substrate 4 of the cell permeability evaluation apparatus 1, monolayer cells can be formed.

(3) Confirmation of tight binding of MBEC4 cells cultured in the permeable substrate 4 of the cell permeability evaluation apparatus 1 [3-1] Confirmation by immunostaining of ZO-1 protein Cell permeability evaluation in this example (1) MBEC4 cells cultured on the permeable substrate 4 of the apparatus 1 were fixed by immersing the permeable substrate 4 together with 4% paraformaldehyde / phosphate buffer (Wako Pure Chemical Industries, Ltd.) for 15 minutes as a primary antibody. Using a ZO-1 protein antibody (Rabbit anti-ZO-1; Invitrogen), which is a tight junction marker, and an anti-rabbit antibody (Goat anti-rabbit IgG; Invitrogen) labeled with Alexa546 as a secondary antibody, a conventional method is used. According to the procedure, immunostaining was performed. Subsequently, Hoechst 33342 (DAIJINDO) was added to a concentration of 1 μg / mL, and the mixture was allowed to stand at room temperature for 15 minutes under light shielding, and then washed with PBS. Thereafter, the fluorescence of Alexa546 and Hoechst33342 was detected using an EM-CCD camera (Hamamatsu Photonics) and a fluorescence microscope (TE2000; Nikon). The result is shown in FIG.

  As shown in FIG. 6, in the MBEC4 cells cultured on the permeable substrate 4, it is clear that the ZO-1 protein indicated by Alexa546 fluorescence exists between the nuclei indicated by Hoechst33342 fluorescence. became. From this result, it was confirmed that MBEC4 cells cultured on the permeable substrate 4 of the cell permeability evaluation apparatus 1 form tight junctions.

[3-2] Confirmation by Measurement of Intermembrane Electrical Resistance (TER) Cell permeability evaluation apparatus 1 in which MBEC4 cells were cultured in Example (1), conventional cell permeability evaluation apparatus 5 in which MBEC4 cells were cultured, and MFLN4 About the conventional cell permeability evaluation apparatus 5 which culture | cultivated the cell, the electrode was put into the area | region of the upper layer side, and the area | region of the lower layer side using MILICELL-ERS (Millipore), and the transmembrane electrical resistance (TER) was measured. . FIG. 7 shows the TER measurement method, and FIG. 8 shows the measurement results.

In the case where a tight bond is formed between cultured cells as shown in FIG. 7 in the permeable substrate 4 of the cell permeability evaluation apparatus 1 or the PET membrane of the conventional cell permeability evaluation apparatus 5, Since electric resistance is generated, the value of TER increases. As a result of the measurement, as shown in FIG. 8, in the conventional cell permeability evaluation apparatus 5 in which MFLN4 cells were cultured, the TER value was about 10 Ω / cm ² , whereas the conventional cells in which MBEC4 cells were cultured A high value of about 210 Ω / cm ² was measured in the permeability evaluation device 5 and about 230 Ω / cm ² in the cell permeability evaluation device 1 in which MBEC4 cells were cultured.

  From these results, the MBEC4 cells cultured on the permeable substrate 4 of the cell permeability evaluation apparatus 1 have the same electrical resistance as the MBEC4 cells cultured on the PET membrane of the conventional cell permeability evaluation apparatus 5. It was confirmed that a high-density tight bond was formed.

<Comparative Example 1> Confirmation of cell-free liposome permeability in PET membrane of conventional cell permeability evaluation device 5 Four types of conventional cell permeability evaluation devices 5 (Millicell; Millipore, Membrane material; PET) with different membrane pore sizes Manufactured, 24 well, membrane pore size; 0.4 μm, 1.0 μm, 3.0 μm, 8.0 μm), the cell-free liposome permeability was calculated by the method described in Example 2. However, the transition time (the time from the addition of the liposome to the upper layer side to the recovery of the Krebs buffer from the lower layer side) was 3 hours, 6 hours, and 9 hours. The result is shown in FIG.

  As shown in FIG. 9, the cell-free liposome permeability is between about 0.17% and about 0.25% at any membrane transition time at a membrane pore size of 0.4 μm, and at a membrane pore size of 1.0 μm. The cell-free liposome permeability is between about 0.23% and about 0.5% when the transition time is 3 hours and 6 hours, and the cell-free liposome permeability when the transition time is 9 hours. Was about 1.75%. On the other hand, at a membrane pore size of 3.0 μm, the cell-free liposome permeability is about 12% when the transition time is 3 hours, and the cell-free liposome permeability is about 17 when the transition time is 6 hours and 9 hours. % To about 18%. Further, when the migration time is 3 hours at a membrane pore size of 8.0 μm, the cell-free liposome permeability is about 19%, and when the migration time is 6 hours and 9 hours, the cell-free liposome permeability is about 9%. % To about 10%.

  From these results, in the conventional cell permeability evaluation apparatus 5 in which no cultured cells are present, when the membrane pore size is 3.0 μm and 8.0 μm, 10% to 20% of liposomes permeate the PET membrane. confirmed. On the other hand, when the membrane pore size was 0.4 μm and 1.0 μm, it was confirmed that most liposomes did not permeate the PET membrane.

Comparative Example 2 Confirmation of MBEC4 Cell Presence Site on PET Membrane (1) Culture of MBEC4 Cells on PET Membrane of Conventional Cell Permeability Evaluation Device 5 Three types of conventional cell permeability evaluation devices with different membrane pore sizes MBEC4 cells were cultured by the method described in Example 3 (1) at 5 (Millicell; Millipore, membrane material: PET, 24 well, membrane pore size: 0.4 μm, 1.0 μm, 8.0 μm).

(2) Confirmation of MBEC4 cell existing site on PET membrane [2-1] Preparation of sample For MBEC4 cells cultured in this comparative example (1), the method described in Example 3 (3) [3-1] Was used for immunostaining of ZO-1 protein. For MBEC4 cells cultured in the conventional cell permeability evaluation apparatus 5 having a membrane pore size of 8.0 μm, nucleation staining using Hoechst 33342 was also performed after immunostaining of ZO-1 protein. A sample was prepared by encapsulating the stained cells together with a membrane in a slide glass using phosphate buffered saline containing 90% (v / v) glycerol.

[2-2] Confirmation of Cell Presence Site by Detection of ZO-1 Protein For the sample prepared in this Comparative Example (2) [2-1], using a confocal laser microscope (LSM510-META; Zeiss) Alexa 546 fluorescence was detected. Detection is performed by changing the observation depth from the upper part of the membrane to the lower part (back side) of the membrane. An image when the observation depth is the smallest is A, and B, C, D, E as the observation depth increases. Images were acquired as, F, G, H, I, J, K, L, M, N, and O. The observation results in the conventional cell permeability evaluation apparatus 5 with a membrane pore diameter of 0.4 μm are shown in FIG. 10, the observation results in the conventional cell permeability evaluation apparatus 5 with a membrane pore diameter of 1.0 μm are shown in FIG. The observation results in the conventional cell permeability evaluation apparatus 5 of 0.0 μm are shown in FIG.

  As shown in FIG. 10, when MBEC4 cells were cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore size of 0.4 μm, Alexa 546 fluorescence was detected in the panels C to L showing the upper part of the membrane, In panels M to O showing the inside of the membrane, Alexa 546 fluorescence was not detected. From this result, it was confirmed that when MBEC4 cells were cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 0.4 μm, monolayer cells could be formed.

  Further, as shown in FIG. 11, when MBEC4 cells are cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 1.0 μm, not only the panels C to I showing the upper part of the membrane but also the inside of the membrane Also in panels J to O, the fluorescence of Alexa546 was detected. From this result, when MBEC4 cells are cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 1.0 μm, monolayer cells cannot be formed, and MBEC4 cells are not only in the upper part of the membrane but also in the pores of the membrane. It was confirmed that it will exist even before.

  Furthermore, as shown in FIG. 12, when MBEC4 cells were cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore size of 8.0 μm, not only panels C to G showing the upper part of the membrane but also the inside of the membrane Fluorescence of Alexa 546 was detected until it reached panels H to J indicating panels and panels K to O indicating the lower part of the membrane. From this result, when MBEC4 cells were cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 8.0 μm, monolayer cells could not be formed, and MBEC4 cells were not only in the upper part of the membrane but also in the pores of the membrane. It has been confirmed that it exists even at the bottom of the membrane.

[2-3] Confirmation of cell existence site by detection of ZO-1 protein and nucleus Subsequently, conventional cell permeability prepared in this comparative example (2) [2-1] and having a membrane pore size of 8.0 μm About the sample of MBEC4 cells cultured on the PET membrane of the evaluation apparatus 5, the fluorescence of Alexa546 and the fluorescence of Hoechst33342 are detected by changing the observation depth by the method described in this comparative example (2) [2-2], Images from A to N were acquired. The result is shown in FIG.

  As shown in FIG. 13, when MBEC4 cells are cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore size of 8.0 μm, not only panels B to H showing the upper part of the membrane but also the inside of the membrane is shown. In panels I to N, Alexa 546 fluorescence and Hoechst 33342 fluorescence were also detected. In particular, in panels I to N, Alexa 546 fluorescence and Hoechst 33342 fluorescence were detected in the membrane pores, and the membrane pores were observed to be clogged with nuclei. From this result, when MBEC4 cells were cultured on a PET membrane of the conventional cell permeability evaluation apparatus 5 having a membrane pore diameter of 8.0 μm, monolayer cells could not be formed, and MBEC4 cells were not only in the upper part of the membrane but also in the pores of the membrane. It was confirmed that it will exist even before.

Comparative Example 3 Confirmation of MBEC4 Cell Presence Site on PET Membrane Coated with Gelatin or Collagen (1) Preparation of Conventional Cell Permeability Evaluation Apparatus 5 Having Coated PET Membrane [1-1] Gelatin coated Preparation of Conventional Cell Permeability Evaluation Apparatus 5 Having PET Membrane Gelatin powder (MP Biomedicals) was added to phosphate buffered saline to 2% (w / w) and autoclaved at 121 ° C. for 10 minutes. To prepare a gelatin solution. A gelatin solution in such an amount that the whole membrane is covered is poured into the upper layer side of a conventional cell permeability evaluation apparatus 5 (Millicell; Millipore, membrane material: PET, 24 wells) having a membrane pore size of 3.0 μm. After incubation at 37 ° C. for 4 hours, the gelatin solution was removed.

[1-2] Production of Conventional Cell Permeability Evaluation Apparatus 5 Having Collagen-Coated PET Membrane Collagen (Nitta Gelatin Co., Ltd.) is added to a diluted hydrochloric acid aqueous solution having a pH of 3.0 so that the concentration becomes 0.3 mg / mL. A solution was prepared. Pour a collagen solution in an amount sufficient to cover the entire membrane in the upper layer area of a conventional cell permeability evaluation apparatus 5 (Millicell; Millipore, membrane material: PET, 24 wells) having a membrane pore size of 3.0 μm. After incubating at room temperature for 1 hour, the collagen solution was removed, and the membrane was washed twice with phosphate buffered saline.

(2) Cell Culture on PET Membrane Coated with Conventional Cell Permeability Evaluation Apparatus 5 Conventional cell permeability evaluation apparatus having a gelatin-coated PET membrane prepared in [1-1] Comparative Example (1) 5 and this comparative example (1) In place of the conventional cell permeability evaluation apparatus 5 having the collagen-coated PET membrane prepared in [1-2], in the cell permeability evaluation apparatus 1, Example 3 (1) MBEC4 cells were cultured by the method described in 1. above.

(3) Confirmation of MBEC4 cell existing site in coated PET membrane For cells cultured in this comparative example (2), immunization with ZO-1 protein was carried out by the method described in comparative example 2 (2) [2-1]. Samples were prepared after staining. Then, the fluorescence of Alexa546 was detected by the method as described in Comparative Example 2 (2) [2-2]. The observation depth was divided into three stages, upper part of membrane, inside of membrane, and lower part of membrane, and images were acquired. The result is shown in FIG.

  As shown in FIG. 14, in any coated PET membrane, not only the fluorescence of Alexa 546 is detected as a whole in the image on the upper part of the membrane, but also in the image inside the membrane, Fluorescence was detected. From these results, when MBEC4 cells were cultured on gelatin-coated PET membrane or collagen-coated PET membrane, monolayer cells could not be formed, and MBEC4 cells existed not only in the upper part of the membrane but also in the pores of the membrane. It was confirmed that

DESCRIPTION OF SYMBOLS 1 Cell permeability evaluation apparatus 2 1st area | region part 3 2nd area | region part 4 Permeability base material 5 Conventional cell permeability evaluation apparatus 6 Upper layer area | region 7 Lower layer area | region 8 Membrane 9 Insert 10 Well plate

Claims (7)

An apparatus for evaluating cell permeability of a polymer compound or a complex thereof,
A first region portion into which a polymer compound or a complex thereof is charged;
A second region portion that is adjacent to the first region portion and contains a polymer compound or a composite thereof that is transmitted from the first region portion; and
Having a permeable substrate disposed on a boundary between the first region portion and the second region portion;
The permeable substrate is composed of fibers made by an electrospinning method using gelatin, and can form a single-layer cell tightly bonded to one surface on the first region side, and is a polymer compound Alternatively, the apparatus is configured to be permeable to the complex.   The apparatus according to claim 1, wherein a diameter D of a fiber produced by electrospinning using gelatin is 500 nm ≦ D ≦ 1000 nm, and a distance I between the fibers is 5 μm ≦ I ≦ 20 μm.   The device according to claim 1 or 2, wherein the polymer compound or the complex thereof is a lipid or a lipid membrane structure. A method for evaluating cell permeability of a polymer compound or a complex thereof,
A step of forming a monolayer cell tightly bonded to one side of a permeable substrate composed of fibers made by an electrospinning method using gelatin and capable of penetrating a polymer compound or a composite thereof. When,
Contacting the monolayer cell with a polymer compound or a complex thereof;
A step of contacting the monolayer cell with a polymer compound or a complex thereof and evaluating cell permeability from the polymer compound or the complex thereof permeated through the monolayer cell and the permeable substrate. Method. A method for evaluating cell permeability of a polymer compound or a complex thereof,
A polymer compound or a composite thereof is brought into contact with one side of a permeable substrate which is composed of fibers made by an electrospinning method using gelatin and is capable of transmitting the polymer compound or the composite thereof. Obtaining a polymer compound or a composite thereof having passed through the permeable substrate,
Forming a monolayer cell tightly bonded to one side of the permeable substrate;
Obtaining a polymer compound or a complex thereof permeating the monolayer cell and the permeable substrate by contacting the monolayer cell with a polymer compound or a complex thereof;
Cell permeability is compared by comparing the obtained polymer compound or a complex thereof permeated through the permeable substrate with the polymer compound or the complex thereof permeated through the obtained monolayer cell and the permeable substrate. And evaluating the method.   The method according to claim 4 or 5, wherein a diameter D of a fiber produced by electrospinning using gelatin is 500 nm ≦ D ≦ 1000 nm, and a distance I between the fibers is 5 μm ≦ I ≦ 20 μm.   The method according to any one of claims 4 to 6, wherein the polymer compound or a complex thereof is a lipid or a lipid membrane structure.

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