BIODEGRADABLE POLYMERIC FILM INCLUDING EXTRACELLULAR MATRIX AND USE THEREOF

Abstract

Provided are a biodegradable polymeric film including an extracellular matrix, and use thereof, and particularly, a method of producing a poly(lactide-co--caprolactone) film including an extracellular matrix, a film produced by the method, and an ophthalmic material including the film.

Claims

1. A method of producing a poly(lactide-co--caprolactone) film comprising an extracellular matrix, the method comprising: producing a two-phase mixture comprising poly(lactide-co--caprolactone) and an extracellular matrix by adding a poly(lactide-co--caprolactone)(PLCL) polymer solution to a solid-phase substrate to which the extracellular matrix (ECM) is attached; and forming a PLCL-ECM film on a surface of the solid-phase substrate by evaporating the solvent from the produced two-phase mixture, wherein the PLCL-ECM film is formed by physical crosslinking between the PLCL and the ECM.

2. The method of claim 1, wherein the ECM is obtained through decellularization of a biological tissue or a cultured cell layer.

3. The method of claim 1, wherein the ECM is obtained through decellularization of a cultured fibroblast cell layer.

4. The method of claim 1, wherein a molecular weight of the PLCL is 100 kDa to 200 kDa.

5. The method of claim 1, wherein the PLCL solution comprises, as the solvent, chloroform, tetrahydrofuran, hexafluoroisopropanol, dimethylformamide, acetone, dimethyl sulfoxide, distilled water, a phosphate buffered solution (PBS), or saline.

6. The method of claim 1, wherein the PLCL solution has a concentration of 0.5% (w/v) to 5% (w/v).

7. The method of claim 1, wherein the evaporating of the solvent is carried out at 5 C. to 60 C.

8. The method of claim 7, wherein the evaporating of the solvent is carried out for 12 hr to 72 hr.

9. The method of claim 1, further comprising separating the PLCL-ECM film from the surface of the solid-phase substrate.

10. A poly(lactide-co--caprolactone) film comprising an extracellular matrix, the film being produced by the method of claim 1.

11. The film of claim 10, wherein the film has a thickness of 5 m to 25 m.

12. The film of claim 10, wherein the film is used as an implant material.

13. A method of regenerating biological tissue, the method comprising administering or implanting, into an individual, a poly(lactide-co--caprolactone) film comprising an extracellular matrix, the film being produced by the method of claim 1.

14. An ophthalmic material comprising the poly(lactide-co--caprolactone) film comprising an extracellular matrix of claim 10.

15. The ophthalmic material of claim 14, wherein the ophthalmic material enhances survival and proliferation of corneal endothelial cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0042] FIG. 1 shows a schematic illustration of a process of producing a poly(lactide-co--caprolactone) film (PLCL-ECM film) including an extracellular matrix according to one exemplary embodiment;

[0043] FIGS. 2A and 2B show physical properties (e.g., transparency) of the PLCL-ECM film according to one exemplary embodiment, wherein FIG. 2A shows results of visual observation of the PLCL-ECM film, and FIG. 2B shows results of examining the thickness of a cross-section of the PLCL-ECM film with a scanning electron microscope;

[0044] FIG. 3 shows results of using a confocal laser microscope to observe the PLCL-ECM film according to one exemplary embodiment immunostained with a fibronectin (FN) antibody, to identify the ECM existing on the PLCL-ECM film;

[0045] FIG. 4 shows results of a live & dead assay to examine the effect of the PLCL-ECM film according to one exemplary embodiment on viability of WI-38 cells;

[0046] FIG. 5 shows results of examining cell attachment through intracellular F-actin and vinculin immunostaining afte seeding human corneal endothelial cells (hCECs) on the PLCL-ECM film according to one exemplary embodiment;

[0047] FIG. 6 shows results of examining the effects of the PLCL-ECM film according to one exemplary embodiment on proliferation of hCEC cells through a CCK-8 assay, as compared with a fibronectin-coated group (PLCL-FN); and

[0048] FIG. 7 shows results of comparing the cell proliferation effects between the PLCL-ECM film according to one exemplary embodiment and a PVA-ECM film, wherein the proliferation of NIH3T3 cells was compared through a CCK-8 assay.

DETAILED DESCRIPTION

[0049] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items. Expressions such as at least one of, when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0050] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1. Production of Poly(Lactide-Co--Caprolactone) Film Including Extracellular Matrix

[0051] 1-1. Preparation of Human Lung Fibroblast-Derived Matrix

[0052] A human lung fibroblast WI-38 cell line (ATCC CCL-75) was seeded at a density of 210.sup.4 cells/cm.sup.2 on a cover slip glass (18 mm). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 l/ml streptomycin was added to the cover slip glass on which the human lung fibroblast WI-38 cell line was seeded, and cultured for about 7 days under general culture conditions (5% CO.sub.2, 37 C.) while replacing the medium every 2 or 3 day. Thereafter, the cultured cells were washed with phosphate-buffered saline (PBS). Subsequently, 0.25% (v/v) Triton-X 100 and 50 mM NH.sub.4OH (Sigma) were added to the washed cells, and then 50 U/ml of DNase I (Invitrogen) and 2.5 l/ml of RNase A (Invitrogen) were added, and decellularization was carried out by incubation at 37 C. for 2 hr. Thereafter, the decellularized extracellular matrix (ECM) was washed with PBS to obtain a human lung fibroblast-derived matrix (hFDM). The obtained human lung fibroblast-derived matrix was used immediately or stored at about 4 C. in the presence of PBS until use.

[0053] 1-2. Preparation of Poly(Lactide-Co--Caprolactone) Solution

[0054] A poly(lactide-co--caprolactone) (PLCL, molecular weight: about 138 kDa, Resormer LC 703, Evonik) copolymer and chloroform (molecular weight: about 119.38, Sigma) were stirred using a magnetic stirrer at 500 rpm for 4 hr, and homogeneously dissolved, and as a result, 2.5% (w/v) of a poly(lactide-co--caprolactone) polymer solution (hereinafter, referred to as a PLCL solution) was prepared using chloroform as a solvent.

[0055] 1-3. Production of Poly(Lactide-Co--Caprolactone) Film Including Extracellular Matrix

[0056] 100 l of 2.5% (w/v) PLCL solution was placed onto the human lung fibroblast-derived matrix prepared in Example 1-1, and exposed at room temperature for 24 hr to fully evaporate the chloroform solvent. During the process, a physical crosslinking reaction between PLCL and human lung fibroblast-derived matrix was induced, and finally, a poly(lactide-co--caprolactone) film including ECM (hereinafter, referred to as a PLCL-ECM film) was developed, as shown in FIG. 1. Thereafter, distilled water was added to the PLCL-ECM film, and left for 5 min. Then, the physically crosslinked PLCL-ECM film was carefully detached from the cover slip glass using a forceps, and transferred to a new plate to reserve a PLCL-ECM film according to one exemplary embodiment.

Example 2. Examination of Physical and Surface Properties of PLCL-ECM Film

[0057] In this exemplary embodiment, physical properties of the PLCL-ECM film produced in Example 1 were examined, and it was also examined whether the human lung fibroblast-derived matrix which is an ECM component was actually attached on the surface of the film. In detail, appearance of the PLCL-ECM film was visually observed. Thereafter, the PLCL-ECM film was cut with a surgical knife, and the cross-section was observed with a scanning electron microscope. Further, the PLCL-ECM film was immunostained using an anti-fibronectin antibody (catalog no. SC-8422, Santa Cruz Biotechnology) as a primary antibody, and Alexa Fluor 488-conjugated anti-mouse IgG antibody as a secondary antibody, and the PLCL-ECM film specifically immunostained with fibronectin was observed with a confocal laser microscope (Zeiss, LSM700).

[0058] As a result, as shown in FIGS. 2A and 2B, the PLCL-ECM film was found to be a transparent film having a thickness of about 10 m. Further, as shown in FIG. 3, it was found that a large amount of the human lung fibroblast-derived matrix was present on the surface of the film separated from the cover slip glass. In other words, these experimental results indicate that the human lung fibroblast-derived matrix present on the surface of the film was attached securely on the PLCL film while well maintaining its original fiber structure, and the polymer film having the above-described physical properties, i.e., thin and transparent properties shows its applicability as an ophthalmic material.

Example 3. Examination of Effect of PLCL-ECM Film on In-Vivo (In Vitro?) Cells

[0059] 3-1. Evaluation of Biocompatibility

[0060] To examine biocompatibility of the PLCL-ECM film, WI-38 cells were dispensed at a density of 110.sup.4 cells/ml on the PLCL-ECM film, and cultured for 24 hr to evaluate viability of the cells by a live & dead assay. In detail, the PLCL-ECM film including WI-38 cells was washed with a Dulbecco's phosphate-buffered saline (DPBS) solution (Sigma-Aldrich), and then co-treated with calcein AM (green) and ethidium bromide (red), and then incubated for 30 min to evaluate cell viability. At this time, live cells were stained green and dead cells were stained red.

[0061] As a result, as shown in FIG. 4, most of WI-38 cells present on the PLCL-ECM film were stained green, and cells stained red were rarely observed. These experimental results indicate that the PLCL-ECM film according to one exemplary embodiment has excellent biocompatibility.

[0062] 3-2. Evaluation of Cell Adhesion Ability

[0063] To examine cell adhesion ability of the PLCL-ECM film, human corneal endothelial cells (hCECs) were dispensed at a density of 110.sup.4 cells/ml on the PLCL-ECM film, and cultured for 24 hr. The PLCL-ECM film including hCECs was washed with a DPBS solution, and then subjected to immunofluorescence staining to examine expression of cell adhesion proteins. In the immunofluorescence staining, the cell nuclei were stained with DAPI (blue), and F-actin and vinculin which are cell adhesion markers were stained with Alexa Fluor 594 (red) and Alexa Fluor 488, respectively.

[0064] As a result, as shown in FIG. 5, both F-actin and vinculin were observed in hCECs cultured on the PLCL-ECM film, consistent with the hCECs distribution on the film as identified via DAPI staining. These experimental results indicate that the PLCL-ECM film according to one exemplary embodiment is able to stably attach human corneal endothelial cells.

[0065] 3-3. Evaluation of Cell Proliferation Ability

[0066] To examine cell proliferation ability on the PLCL-ECM film, cultured hCECs were subjected to a CCK-8 assay. In detail, the PLCL-ECM film including hCECs at a density of 110.sup.4 cells/ml was washed with a DPBS solution, and then 500 l of medium was added thereto, and 50 l of water-soluble tetrazolium salt-8 (WST-8) was added to the cell-dispensed solution. Thereafter, cells were incubated for 2 hr in the dark, and then absorbance at 450 nm was measured by an ELISA reader. The measurement was expressed as a cell proliferation rate (%), based on a level of the cells present on the fibronectin (FN)-coated PLCL film (PLCL-FN) on day 0, and the above measurement was performed on day 2 and 5 after culture. As a control, an FN-coated PLCL film (PLCL-FN) was used.

[0067] As a result, as shown in FIG. 6, the PLCL-ECM film was able to improve the proliferation ability of hCECs over time, and in particular, the cell proliferation level in the PLCL-ECM film was significantly improved, as compared with the control.

Example 4. Comparison of Cell Proliferation Effects According to Polymer Materials

[0068] In this exemplary embodiment, cell proliferation effects were compared between the PLCL-ECM film according to one exemplary embodiment and a PVA-ECM film based on a polyvinyl alcohol (PVA) material. The PVA-ECM was produced in the same manner as in Example 1, and a CCK-8 assay was carried out using NIH3T3 cells in the same manner as in Example 3-3. Meanwhile, the measurement was expressed as a cell proliferation rate (%), based on a level of the cells present on the PVA-ECM film on day 0.

[0069] As a result, as shown in FIG. 7, the cell proliferation level in the PLCL-ECM film was significantly improved, as compared with the PVA-ECM film, and in particular, on day 5 after culture, the cell levels of the films showed a remarkable difference of more than twice.

[0070] A method according to an aspect may provide a polymer film having excellent biocompatibility and biological efficacy through a physical crosslinking reaction between poly(lactide-co--caprolactone) and an extracellular matrix.

[0071] The poly(lactide-co--caprolactone) film including an extracellular matrix according to an aspect may exhibit excellent cell adhesion ability and may also remarkably improve cell proliferation ability, thereby being applied to biological materials including an ophthalmic material in various fields.

[0072] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.