Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/08—Materials for coatings
  • A61L31/082—Inorganic materials
  • A61L31/084—Carbon; Graphite
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
  • A61L29/08—Materials for coatings
  • A61L29/10—Inorganic materials
  • A61L29/103—Carbon

Definitions

• the invention relates to graphene coating on endovascular stents as an endothelialization enhancement and restenosis reduction layer and an implantable medical device with such coating.
• the prior art solution disclosed in the International Patent Application No. WO2013109891A1 relates to a barrier product comprising a substrate and a continuous layer based on graphene that adheres to the substrate, wherein the continuous layer is substantially impermeable to small molecules.
• the product is an implant, and the layer based on graphene includes functionalized graphene, graphene oxide or reduced graphene oxide.
• the document also discloses a method for the production of the barrier product, involving contacting the product with liquid solution of graphene flakes, a method for coating a substrate impermeable to small molecules, a method for sealing holes in a substrate, a barrier layer containing flat sheets of graphene oxide.
• the continuous graphene-based coating is composed of graphene plates (lateral dimension between 0.1 ⁇ and 5 ⁇ ) arranged planarly and overlapping one another layer by layer, where the thickness of graphene layer is between 5 nm and 200 nm.
• the aim of the present invention is a novel use of graphene coating for coating implantable medical devices to enhance endothelialization and reduce restenosis as well as to improve the biocompatibility of implantable medical devices with the implantation site.
• the invention relates to the use of graphene coating for coating an implantable medical device, characterized in that the graphene coating consists of at least one graphene layer.
• the graphene layer forms a uniform graphene coating comprised of overlapping graphene flakes that form a compact coating, completely coating the surface of the coated implantable medical device.
• the graphene coating is comprised of between 1 and 10 non-continuous graphene layers, more preferably one non- continuous graphene layer.
• Another object of the invention is an implantable medical device characterized in that it comprises at least one coating comprised of at least one non-continuous graphene layer on at least part of its surface.
• the implantable medical device of the present invention is made of metal or plastic.
• the implantable medical device of the present invention is a stent or a catheter, more preferably a stent for coronary, peripheral, carotid, renal arteries.
• a graphene coating in the form of at least one non-continuous layer of graphene for coating an implantable medical device solves the problem of a long- term process of endothelial regeneration after implantation and reduces the risk of restenosis.
• graphene coating in the form of at least one non-continuous layer of graphene for coating an implantable medical device, including stents, results in an enhanced adhesion of the endothelial cells to the implanted medical device, increased endothelial cell growth at the implantation site as well as inhibits the process of endothelial differentiation into fibroblasts, thus reducing the risk of restenosis at the site of implantation of a medical device, stent restenosis in particular. Additionally, the graphene coating protects the implant against corrosion.
• the use of graphene coating of the present invention allows for a significant reduction of the regeneration period after implantation of a medical device coated with the graphene coating of the present invention as well as for a limited use of anticoagulation therapy. Furthermore, it was observed that in the case of implantable medical devices of the present invention, the biocompatibility of the device with the implantation site is improved, which reduces the risk of rejection.
• Embodiments of the present invention are of major importance in clinical practice as the use of implantable medical devices comprising at least one coating comprised of at least one non-continuous graphene layer allows for a significant reduction of postoperative complications, reduction of the regeneration period after the implantation and for a prolonged use of implantable medical devices, stents in particular, which results in improved comfort of patients after implantation.
• Fig.l. shows representative images of coronary artery endothelial cells labeled with ab 15605 (endothelial cell- specific marker), after 72 hrs of culture on steel plates and plates coated with a graphene layer.
• the group denoted as 1-5 and S2-S3 refers to steel plates whose surface was hydrophilized before applying a layer of graphene; the P1-P3 group of plates refers to non- hydrophilized steel plates, onto which graphene was applied; n- the number of plates in a given group.
• Fig. 3 shows representative images of FDA-labeled cells after 72 hrs of culture on steel plates and graphene-coated plates. The test was performed on three groups of graphene plates (1-5).
• Fig. 6 shows representative images of coronary artery endothelial cells labeled with ab 15605 (the red channel, endothelial cell specific marker) and ab28244 (green channel, fibroblast specific marker) as well as an image of overlapped channels after 72 hrs of culture on steel plates and graphene-coated plates.
• B study group 1-5.
• the cell culture used in the experiments was kept under standard conditions, i.e. at 37°C, in 5% C0 2 enriched atmosphere saturated with water vapor.
• the complete culture medium was the Vascular Cell Basal Media (ATCC) supplemented with Endothelial Cell Growth Kit-BBE (ATCC) comprising: bovine serum (2% (v/v), ATCC), L-glutamine (10 mM, ATCC), bovine brain extract (0.2% (v/v), ATCC), rhEGF (5 ng/mL, ATCC), heparin sulfate (0.75 U/mL, ATCC), hydrocortisone hemisuccinate (1 ⁇ g/mL, ATCC), ascorbic acid (50 mg/mL, ATCC), gentamicin (10 mg/mL, Sigma Aldrich), amphotericin B (25.25 mg/mL, Sigma Aldrich), streptomycin (10 ⁇ g/mL, Sigma Aldrich) and penicillin (10 U/mL, Sigma Ald
• the cells were passaged 1:3 after reaching 80% confluence. For this purpose, the cells were harvested from the substrate using trypsin (ATTC) after previous medium collection. The reaction was inhibited after adding the complete medium. Cellular viability was assessed using trypan blue exclusion. Endothelial cell cultures with more than 90% viability were used in experiments.
• the samples were disinfected by washing in 70% (v/v) ethanol, followed by washing with PBS three times (Sigma Aldrich).
• the disinfected and washed discs were placed on 24-well culture plates for adherent cell culture (Greiner Bio-One, Cellstar). Then, they were loaded onto 500 ⁇ . of endothelial cell suspension (Human Primary Coronary Artery Endothelial Cells, ATCC) to achieve optimal cell density of 1 x 104/cm 2 in a well. Control cultures (cell culture on a plate without the disc) were also included each time. Cells were cultured for 72 hrs, until the below described tests.
• the group of plates denoted as 1-5 and S2-S3 refers to steel plates whose surface was hydrophilized before applying a layer of graphene; the P1-P3 group of plates refers to non- hydrophilized steel plates, onto which graphene layers were applied; n- the number of plates in a given group.
• Indirect immunofluorescence was used to identify coronary artery endothelial cells on the plates.
• Antibodies against endothelial cells [BW 200] ab 15605, Abeam), which, after binding to the plated cells, were incubated with antibodies conjugated with DyLight 550 Fluorochrome emitting red fluorescence, were used.
• a culture plate was transferred under a laminar chamber, where the cultures were discontinued by decanting the culture medium, washing the cells 3x with PBS, followed by fixing the cells by 15-minute incubation in 4% (w/v) formaldehyde in PBS at room temperature. Then, the fixative was decanted, the cells were washed 3x with PBS, followed by cell incubation in a blocking buffer: 1% BSA (w/v), 10% (v/v) goat serum, 3M glycine in 0.1% (v/v) Tween-PBS, pH of 7.5, for 1 hour at room temperature.
• the blocking buffer was decanted, washed 3x in PBS and the cells were stained overnight in a humid chamber at 4°C in a solution of primary antibodies (mouse monoclonal antibodies against human endothelial cells [BW 200], ab 15605, Abeam, at 1 ug/mL in 1% (w/v) BSA in PBS).
• Primary antibodies mouse monoclonal antibodies against human endothelial cells [BW 200], ab 15605, Abeam, at 1 ug/mL in 1% (w/v) BSA in PBS.
• Antibody- coated plates were washed 3x with PBS, then incubated for 45 minutes at room temperature, in a dark and humid chamber in a solution of primary antibodies: polyclonal goat anti-mouse IgG H&L labeled with DyLight 550, ab96872, Abeam diluted 1:500 in 0.2% (w/v) BSA in PBS.
• the images were taken using a camera (Canon, EOS 650D) coupled to a program for image analysis (Canon Utilities EOS Capture). Quantitative image analysis was performed using Image J (version 1,50b 14).
• fluorescein diacetate in a cell is possible only when the cell membrane is intact and the cell contains active enzymes, including esterases. Their activity results in the deesterification of fluorescein.
• a culture plate was transferred under a laminar chamber, where the cultures were discontinued by decanting the culture medium, washing the cells 3x with PBS, and then fluorescein diacetate (FDA, 10 ⁇ ) was applied on the plate and the cells were incubated for 15 minutes in a dark and humid chamber at 37°C.
• the cells were evaluated with a fluorescence microscope (Nikon, ECLIPSE E600) at 200x magnification. Cells showing a positive reaction (the presence of green fluorescence) were counted on the individual study and control plates.
• the viability of the cells present on the discs was calculated by adequately comparing the total number of labeled cells obtained for the individual study discs with the total number of labeled cells present on control plates, assuming that the number of labeled cells present on control plates represented 100% (Fig. 3, Fig.4).
• This method uses an anionic dye - sulforhodamine B, which, under appropriate pH conditions, shows the ability to electrostatically bind proteins.
• the quantity of proteins equivalent to sulforhodamine absorption is a direct indicator of the number of cells in culture.
• a commercially available kit was used for the test: In Vitro Toxicology Assay Kit Sulforhodamine B based (Sigma Aldrich). Performing the test:
• the study plate was transferred under a laminar chamber, where the cultures were discontinued by fixing the cells for 1 h at 4°C in cold 50% (w/v) trichloroacetic acid (TCA, Sigma Aldrich). The final TCA concentration in a well was 10% (w/v). The plates with fixed cells were washed five times with distilled water and allowed to air dry for 10 minutes.
• TCA trichloroacetic acid
• the cells fixed on the discs were stained using 250 ⁇ ⁇ of 0.4% (w/v) sulforhodamine B solution (SRB, Sigma Aldrich) in 1% acetic acid. The cells were stained for 30 minutes at room temperature. Unbound SRB was removed by washing the wells five times with 1% (v/v) acetic acid. After re-drying, 500 ⁇ ⁇ portions of 10 mM unbuffered Tris (Sigma Aldrich) were added to wells, followed by 10-minute shaking to dissolve SRB.
• SRB sulforhodamine B solution
• Proliferation of the cells present on the study discs was calculated by adequately comparing the absorbance values obtained for the individual study discs with the absorbance values for control plates, assuming that the absorbance of control plates represented 100% (Fig. 5). Absorbance for the complete culture medium represented a blind test.
• Indirect immunofluorescence method was used for the assessment of the type of cells covering study plates: coronary artery endothelial cells/fibroblasts.
• the cells were labeled with antibodies showing fluorescence in a red channel (anti-endothelial cell antibodies [BW-200]), whereas cells showing features of differentiation into fibroblasts were labeled with antibodies emitting light in the green channel (antibodies against fibroblast activation protein alpha (FAP-a)).
• a culture plate was transferred under a laminar chamber, where the cultures were discontinued by decanting the culture medium, washing the cells 3x with PBS, which was followed by fixing the cells by 15-minute incubation in 4% (w/v) formaldehyde in PBS at room temperature. Then, the fixative was decanted, the cells were washed 3x with PBS and then incubated in a blocking buffer: 1% BSA (w/v), 10% (v/v) donkey serum 10% (v/v) goat serum, 3M glycine in 0.1% (v/v) Tween-PBS, pH 7.5, for 1 h at room temperature. The blocking buffer was decanted, washed 3x with PBS, and the cells were stained overnight in a humid chamber at 4°C in primary antibody solution:
• FAP Rabbit anti-Human Polyclonal antibodies (fibroblast activation protein alpha), ab28244 Abeam, at a concentration of 10 ug/mL in 1% (w/v) BSA in PBS.
• DyLight 488 excitation light: blue (max 493 nm), emission - green light (518 nm).
• DyLight 550 excitation light: green (max 562 nm), emission - red light (576 nm).
• the images were taken using a camera (Canon, EOS 650D) coupled to a program for image analysis (Canon Utilities EOS Capture). Quantitative image analysis was performed using Image J (version 1,50b 14).

Landscapes

• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Inorganic Chemistry (AREA)
• Epidemiology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Heart & Thoracic Surgery (AREA)
• Surgery (AREA)
• Vascular Medicine (AREA)
• Materials For Medical Uses (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=57190212

Family Applications (1)

Country Status (3)

Families Citing this family (3)

Family Cites Families (2)

• 2015
  • 2015-10-14 PL PL414368A patent/PL414368A1/pl unknown
• 2016
  • 2016-10-12 EP EP16785274.8A patent/EP3362108A1/de not_active Withdrawn
  • 2016-10-12 WO PCT/PL2016/050048 patent/WO2017065625A1/en not_active Ceased

Also Published As

Similar Documents

Legal Events