Bioactive nanofiber cyto-scaffold

Abstract

The invention relates to obtaining nanofibers that contain biocompatible polymers and using the product obtained by making them bioactive through linking covalent proteins to said nanofibers in tissue engineering.

Claims

1. A bioactive nanofiber comprising a poly(m-anthranilic acid) (P3ANA), a poly(caprolactone) (PCL) and an arginine-glycine-aspartic acid (RGD) peptide.

2. An electrospinning method for producing a bioactive nanofiber, comprising the following steps: dissolving 15% by weight of PCL (poly(caprolactone)) in a tetrahydrofuran:dimethylformamide (THF/DMF) solution, wherein the volume ratio of THF to DMF is 1:1, obtaining electro spun solution by adding 15% by weight of P3ANA (poly(m-anthranilic acid)) with respect to PCL into the PCL solution to obtain a PCL/P3ANA solution, loading the PCL/P3ANA solution into a syringe, placing the syringe into an electrospinning device, applying a voltage between 10-20 kV to the PCL/P3ANA solution in the syringe, setting a feed rate of the PCL/P3ANA solution to obtain a P3ANA/PCL nanofiber onto a collector, covalently immobilizing RGD (arginine-glycine-aspartic acid) peptide to the P3ANA/PCL nanofiber by carbodiimide binding reaction, to obtain a nanofiber mat, freshly preparing an EDC/NHS solution comprising an 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and an N-hydroxysuccinimide (NHS) before a reaction in a cold 0.1 M 2-(n-morpholino) ethansulfonic acid (MES) in one to one molar proportion, activating the nanofiber mat with the EDC/NHS solution by shaken incubation for 1.5 hours to 2.5 hours at room temperature between 100 rpm and 300 rpm to obtain activated nanofiber mats, washing the activated nanofiber mats twice by shaking with MES buffer for 5-15 minutes and between 100-300 rpm to obtain washed nanofibers, activating the washed nanofibers by shaken incubation in the MES buffer containing RGD peptide for 1.5 hours to 2.5 hours at room temperature between 100 rpm and 300 rpm to obtain an activated washed nanofiber, and washing a nanofiber mat surface of the activated washed nanofiber twice by shaking with MES buffer for 5-15 minutes and between 100-300 rpm for removing RGD peptide molecules physically attached to the nanofiber mat surface.

3. The method according to claim 2, wherein a distance between a needle tip of the syringe and the collector is maintained at a fixed distance.

4. The method according to claim 2, wherein a distance between the needle tip of the syringe and the collector is 15 cm.

5. The method according to claim 2, wherein the feed rate of the PCL/P3ANA solution is set to 1 mL/h.

6. The method according to claim 2, wherein the prepared nanofiber mats are activated by being shaken and incubated with the EDC/NHS solution for 2 hours at room temperature, 200 rpm.

7. The method according to claim 2, wherein the activated nanofiber mats are washed with MES buffer twice by shaking at 200 rpm, 10 minutes respectively.

8. The method according to claim 2, wherein the washed nanofibers are activated by being shaken at 200 rpm and incubated in the MES buffer comprising RGD peptide for 2 hours at room temperature.

9. The method according to claim 2, wherein the nanofiber mat surface is washed by being shaked with the MES buffer twice at 200 rpm for 10 minutes respectively to remove the RGD peptide molecules physically attached to the nanofiber mat surface.

10. The method according to claim 2, wherein a 15 kV voltage is applied to the PCL/P3ANA solution in the syringe.

11. An RGD peptide immobilized PCL/P3ANA bioactive nanofiber obtained by the method according to claim 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Fabrication of PCL/P3ANA nanofibers by electrospinning method.

(2) FIG. 2: Covalent binding of RGD peptide to the nanofibers by EDC-NHS method.

(3) FIG. 3: FTIR-ATR spectra of PCL, PCL/3ANA nanofibers (A) and RGD peptide bound nanofibers (B).

(4) FIG. 4: EIS measurement schematic view (top), Nyquist plot of RGD peptide bound PCL/P3ANA nanofibers.

(5) FIG. 5: SEM view of cells being grown on PCL/P3ANA-RGD nanofibers.

REFERENCE NUMERALS

(6) 1. Syringe Pump 2. High voltage source 3. Nanofiber mat.

DETAILED DESCRIPTION

(7) In the bioactive nanofiber cyto scaffold of the invention, PCL/P3ANA nanofibers are produced by electrospinning method comprising the following steps:

(8) First, dissolving 15% by weight poly(caprolactone) (PCL) into a tetrahydrofuran/dimethylformamide (THF/DMF, 1:1 by volume) solution.

(9) Obtaining an electro spun solution by adding 15% by weight P3ANA to the PCL solution with respect to the previously synthesized PCL.

(10) Loading the PCL/P3ANA solution into a syringe, the syringe is preferably a 23 G needle, 5 ml volume syringe having an outer diameter of 0.7 mm.

(11) Placing the syringe containing the PCL/P3ANA solution into a electrospinning device and connected to the high voltage direct current (DC) source.

(12) Applying a 10-20 kV voltage to the polymer solution and said voltage is preferably 15 kV.

(13) Wherein a distance between a needle tip of the syringe and a collector is kept fixed and said distance is preferably 15 cm.

(14) Feed rate of the solution is set to 1 mL/h by a syringe pump.

(15) Covalently immobilizing the RGD peptide is to the P3ANA/PCL nanofibers by using carbodiimide binding reaction.

(16) Fresh preparing agents comprising an 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and an N-hydroxysuccinimide (NHS) just before the reaction in cold a 0.1 M 2-(n-morpholino) ethansulfonic acid (MES) in one to one molar proportion and preferably in 10 mg/ml concentration.

(17) Activating the prepared nanofiber mat by shaken incubation with the EDC/NHS solution for 1.5 to 2.5 hours, preferably for 2 hours at room temperature between 100 rpm and 300 rpm, preferably at 200 rpm.

(18) Washing the activated nanofiber mat by shaking with the MES buffer for 5-15 minutes, preferably for 10 minutes and by between 100-300 rpm, preferably at 200 rpm.

(19) Activating the washed nanofibers by shaken incubation in the MES buffer containing the RGD peptide for 1.5-2.5 hours, preferably for 2 hours at room temperature between 100-300 rpm, preferably at 200 rpm.

(20) Nanofiber mat surface is washed twice by shaking with MES buffer for 5-15 minutes, preferably for 10 minutes and between 100-300 rpm, preferably at 200 rpm for removing the RGD peptide molecules that may physically attach to the surface.

(21) PCL and P3ANA ratio in the composition of the nanofiber of the invention can be changed to allow nanofiber yield by electrospinning method. ITO-PET provides physical support to the PCL/P3ANA nanofibers. The nanofibers are yielded on the ITO-PET to enable reproducibility of the electrochemical measurements. During the cell experiments, the nanofibers on the ITO-PET or just the nanofibers can be used. PCL/P3AAN nanofibers can serve as cytoscaffold. Instead of ITO-PET, the nanofiber can be obtained on a different support material such as glass, FTO-glass (fluorine doped tin oxide coated glass) or ITO-glass.

(22) Bioactivation of the nanofibers by RGD peptide is proved through electrochemical impedance spectroscopy. Electrochemical impedance spectroscopy measurements are performed by an electrochemical measurement device (potentiostat) and triple electrode system is used in the measurements. In this triple electrode system; the product of the invention (PCL/P3ANA-RGD) is used as the working electrode, silver (ag) wire is used as reference electrode and platinum (Pt) electrode is used as the counter electrode. EIS measurements are performed at room temperature in 0.1M phosphate buffer (PBS) with a pH value of 7.4 between 0.01 Hz and 100 kHz frequency range and by applying 10 mV alternative current.

Embodiment 1: Yield of PCL/P3ANA Nanofibers on ITO-PET by Electrospinning Method

(23) PCL/P3ANA polymer solution is loaded into the syringe (outer diameter 0.7 mm, with 23 G needle) and the nanofibers are obtained by applying 15 kV DC voltage at 1 mL/hour feed rate on the aggregator placed 15 cm away. In order to provide physical support to the nanofiber mat and to increase the strength of the obtained electrode, the nanofibers are aggregated on the semi-conductive ITO-PET (Indium tin oxide coated Polyethylene terephthalate film).

Embodiment 2: Covalent Binding of the RGD Peptide on the PCL/P3ANA Nanofibers

(24) Carboxyl groups contained within the structure of the nanofibers incubated in the 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) solution is activated. One double bond of the Carbodiimide (EDC) chemical is added to the OH group in the carboxyl group present in the nanofiber structure and O-acylurea product is formed then this product is converted to succinimide ester (COOSuc) product in the presence of NHS. Succinimidylesterreacts with the primer amine (NH.sub.2) in the RGD peptide structure and RGD peptide binds to the nanofibers covalently.

Embodiment 3: FTIR-ATR Characterization of PCL, PCL/P3ANA (FIG. 3A) and RGD Bound Nanofibers (FIG. 3B)

(25) In FIG. 3A, the peak value of the CO groups in the PCL nanofibers is seen at 1730 cm.sup.1. After addition of P3ANA to the nanofiber structure new peaks can be seen at 1690 cm.sup.1, 1580 cm.sup.1 and 1510 cm.sup.1 belonging to P3ANA respectively as CO stretching, CC stretching and NH stretching which are not present in the PCL structure. In FIG. 3B, FTIR-ATR graph obtained after binding of the RGD peptide to the nanofiber is shown. In the FTIR spectrum, 1700-1600 cm.sup.1 is a region sensitive to the protein structure. The peak seen at 1560 cm.sup.1 in the FTIR spectrum represents the NH bond in the RGD structure. Moreover, the peaks seen at 1740 cm.sup.1, 1650 cm.sup.1 and 1540 cm.sup.1 belong respectively to the CO stretching in the ester group, Amid I CO stretching and NH deformation in Amid II in the RGD structure.

Embodiment 4: EIS Measurements and Analysis Related to RGD Peptide Bound PCL/P3ANA Nanofibers

(26) During EIS measurement triple electrode system (PCL/P3ANA or PCL/P3ANA-RGD nanofibers produced in the invention as working electrode, silver (Ag) wire as reference electrode and platinum (Pt) electrode as the counter electrode) is used (FIG. 4top). In the Nyquist graph of the PCL/P3ANA nanofibers, a significant difference is seen after binding of the RGD peptide to the nanofibers. The radius obtained by the points where the semi-circle obtained from the Nyquist graph given in FIG. 4 (bottom) intersects the x axis represents the charge transfer resistance (Rct) and the linear part represents the capacitive behavior. While PCL/P3ANA nanofibers have a linear Nyquist graph, semi-circular curves are obtained in Nyquist graphs obtained after covalent binding of the RGD peptide. This situation suggests that RGD peptide increases the resistance of the nanofibers. This increase in the charge transfer resistance is described by formation of a layer that is blocking ion transfer on the nanofiber surface by the RGD peptide molecules.

Embodiment 5: SEM Image of Cells Grown on PCL/P3ANA-RGD Nanofibers

(27) The cells are grown on the nanofiber for 9 days and distribution and growth of the cells on the nanofiber is shown by SEM. At the end of 9th days, the cells are spread on the nanofiber surface and grown. SEM image shows that PCL/P3ANA-RGD nanofibers are biocompatible and promotes cell reproduction.