Process for a preparation of the modified porcine plasma fibronectin for enhanced wound healing

Abstract

This invention reveals the potential applications of modified porcine plasma fibronectin that could be applied as a safe material for clinical wound healing and tissue repair. In order to seek safe sources of plasma fibronectin for practical consideration in wound dressing, this invention isolated and modified fibronectin from porcine plasma and demonstrated that modified porcine plasma fibronectin has similar ability as homo plasma fibronectin being as a suitable substrate for stimulation of cell adhesion and directed cell migration. The present invention also reveals a material and a pharmaceutical composition enhance wound healing.

Claims

1. A pharmaceutical composition for enhanced wound healing in a subject, wherein the pharmaceutical composition comprises: a) a modified porcine fibronectin, b) a collagen, and c) a hyaluronic acid or a pharmaceutically acceptable salt thereof; wherein the modified porcine fibronectin was produced by a method comprising treating porcine fibronectin with an enzyme composition that cleaves the glycans attached to the fibronectin protein, and digesting the porcine fibronectin into digested porcine fibronectin fragments; wherein the cleaved glycans are a plurality of sialic acid molecules, wherein the plurality of sialic acid molecules comprises N-acetylneuraminic acid (Neu5Ac) and/or N-glycolylneuraminic acid (Neu5GC) residues, wherein >80% of the plurality of sialic acid molecules are removed.

2. The pharmaceutical composition according to claim 1, wherein the enzyme composition comprises α2-3,6,8 Neuraminidase.

3. The pharmaceutical composition according to claim 2, wherein the enzyme composition further comprises a proteinase with ability to digest fibronectin.

4. The pharmaceutical composition according to claim 3, wherein the proteinase comprises matrix metalloproteinase 3.

5. The pharmaceutical composition according to claim 1, wherein the porcine fibronectin is obtained by a step comprising isolating porcine fibronectin on a size exclusion column with a buffer under conditions that preserve the attachment of the glycans to the fibronectin protein prior to the step of treating porcine fibronectin with an enzyme composition that cleaves the glycans attached to the fibronectin protein.

6. A material for enhanced wound healing in a subject, the material comprises a modified porcine fibronectin, wherein the modified porcine fibronectin was produced by a method comprising treating porcine fibronectin with an enzyme composition that cleaves the glycans attached to the fibronectin protein, and digesting porcine fibronectin into digested porcine fibronectin fragments; wherein the cleaved glycans are a plurality of sialic acid molecules; wherein >80% of the plurality of sialic acid molecules are removed.

7. The material according to claim 6, wherein the enzyme composition further comprises a proteinase with ability to digest fibronectin.

8. The material according to claim 7, wherein the proteinase comprises the matrix metalloproteinase 3.

9. The material according to claim 6, wherein the enzyme composition comprises α2- 3,6,8-neuraminidase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the purification method.

(2) FIG. 2 shows the fractions of the washed materials and eluted materials that obtained from the gelatin-Sepharose Fast Flow 4B column.

(3) FIG. 3 shows the percentage of the spectral counts of the glycans with or without sialic acids (Neu5Ac or Neu5GC) relative to the total spectral counts for individual detected N-glycosylation sites.

(4) FIG. 4a-c shows the isolated homo and porcine fibronectin proteins exhibited similar wound closure effect using U2OS, HFF1 and Hela cells.

(5) FIG. 5 shows U2OS cells were plated on coverslips coated with the indicated fibronectin concentration (μg/ml) for 30 min and then the images were taken by phase contrast microscopy. Bar, 100 μm.

(6) FIG. 6 shows the area of cells spreading on coverslips coated with the indicated fibronectin concentration (μg/ml).

(7) FIG. 7 shows U2OS cells were plated on the indicated concentration of fibronectin for 30 min and then their cell attachment was measured. Fold of cells remaining attached on the indicated concentration of fibronectin relative to that on 0 μg/ml fibronectin.

(8) FIG. 8 shows TIRFM images of U2OS cells that had been plated for 1.5 h on coverslips coated with the indicated fibronectin concentration and immunostained with paxillin. Bar, 10 μm.

(9) FIG. 9 shows the sum of the total area of paxillin-marked focal adhesions within a cell versus the fibronectin concentration.

(10) FIG. 10 shows the removal of sialic acids from porcine fibronectin did not affect the cell-fibronectin association.

(11) FIG. 11 shows the MMP3 digestion of porcine fibronectin result.

(12) FIG. 12 shows percentage of wound closure by treating with porcine fibronectin or porcine fibronectin with MMP3.

DETAILED DESCRIPTION OF THE INVENTION

(13) In order to seek safe sources of plasma fibronectin for practical consideration in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that porcine plasma fibronectin has similar ability as homo plasma fibronectin being as a suitable substrate for stimulation of cell adhesion and directed cell migration.

(14) This invention further defined N-glycosylation sites and N-glycans on homo and porcine plasma fibronectin. These N-glycosylation modifications on plasma fibronectin that synergistically support integrin-mediated signals are necessary and sufficient in mediating cellular adhesion and directed cell migration. Our study not only determines the important function of N-glycans on both homo and porcine plasma fibronectin mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin that could be applied as a material for clinical wound healing and tissue repair.

Example 1. Materials and Cells

(15) U2OS (human bone osteosarcoma cell line) and Hela (human cervical adenocarcinoma epithelial cell line) were gifts from Prof. R.-H. Chen's laboratory (Academia Sinica, Taipei, Taiwan) and were maintained in DMEM-high glucose (Invitrogen) supplemented with 10% FBS (Invitrogen) and 1% antibiotic solution (penicillin and streptomycin; Invitrogen) under 5% CO2. HFF1 (human foreskin fibroblasts) cells were purchased from ATCC and were maintained in DMEM-high glucose supplemented with 15% FBS and 1% antibiotic solution (penicillin and streptomycin) under 5% CO2. The homo plasma was obtained from human blood donated by blood donors. All methods related to human blood were carried out in accordance with relevant guidelines and regulations. All experiments protocols related to human blood were approved by the Ethics Committee of the Institutional Review Board (IRB) of National Yang-Ming University. Informed consent was obtained from all subjects. The porcine plasma was obtained from CHAISHAN FOODS CO., LTD.

Example 2. Plasma Fibronectin Preparation Procedure

(16) Plasma Fibronectin Purification

(17) This invent provides a method for purify the glycosylation fibronectin from porcine, comprising: step 1, cleared plasma was passed through a pre-column of gelatin-Sepharose Fast Flow 4B; step 2, removing nonspecifically adsorbed proteins to the gel with sequential washing with TBS-EDTA 50 ml; step 3, removing nonspecifically adsorbed proteins to the gel with sequential washing with 1 M NaCl 50 ml; step 4, removing nonspecifically adsorbed proteins to the gel with sequential washing with <0.5 M Arginine (Arg) 50 ml; step 5, eluting fibronectin sample with >0.5M Arg; step 6, dialyzing fibronectin sample with TBS (pH 5-8) for 24 hours at 4° C.; step 7, concentrating fibronectin sample by Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa).

(18) Result

(19) This invention used a plasma fibronectin purification method to isolate high quality fibronectin proteins with glycans preservation from porcine plasma. Plasma was loaded into a pre-column of gelatin-Sepharose Fast Flow 4B. The gel was washed sequentially with TBS-EDTA, 1 M NaCl and <0.5 M Arginine (Arg) to remove non-specific binding proteins, leaving bound fibronectin for elution with >0.5 M Arg. The fractions of eluted fibronectin were pooled and dialyzed in TBS for 48 h at 4° C. and concentrated by Vivaspin 20 centrifugal concentrator (Molecular Weight Cut Off: 100 kDa) (FIG. 1-2).

(20) Plasma Fibronectin Modification

(21) This invent provides a method to modify the glycosylated porcine fibronectin, comprising: step 1, preparing porcine plasma fibronectin (1 mg) in buffer (pH 5˜7); step 2, adding 5˜50 units α2-3,6,8 Neuraminidase (One unit is defined as the amount of enzyme required to cleave >95% of the terminal α-Neu5Ac from 1 nmol Neu5Acα2-3 Galβ1-3 GlcNAcβ1-3 Galβ1-4Glc-7-amino-4-methyl-coumarin (AMC), in 5 minutes at 37° C. in a total reaction volume of 10 μl); step 3, incubate for 1˜24 hours in 37° C.

(22) Glycopeptide Identification for Plasma Fibronectin

(23) Precipitated fibronectin protein pellets (˜10 □g) were subjected to protein digestion using a protocol with trypsin. The digested peptide mixtures were dissolved in 0.1% formic acid and then analyzed using a Dionex Ultimate 3000 nanoLC system (Thermo Scientific) interfaced to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) equipped with a PicoView nanosprayer (New Objective). The peptides were loaded directly onto a 25 cm×75 □m C18 column (Acclaim PepMap® RSLC, Thermo Scientific) and separated using a 120-min linear gradient of 100% mobile phase A (0.1% formic acid in water) to 40% mobile phase B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nL/min. The eluted peptides were detected in the positive ion mode using a nanospray source. The mass spectrometer was programmed in the data-dependent mode over 3 secs, which consisted of a cycle of one full-scan mass spectrum (400-2000 m/z) on the Orbitrap scan with 120,000 resolution at m/z 400 and an automatic gain control (AGC) target at 200,000 followed by quadrupole isolation with higherenergy collisional dissociation (HCD) MS2 at a normalized collision energy of 30%. HCD MS2 fragment ions detected in the Orbitrap analyzer at 30,000 resolution (AGC target at 100,000) with any previously selected ions dynamically excluded for 60 s. For the database search, the MS datasets for homo and porcine plasma fibronectin were performed using the Byonic™ search energy (Protein Metrics, v.2.7.4) against FN1 human or FN1_Sus scrofa from the Swiss Prot (Swiss Institute of Bioinformatics) database, respectively. Protein modifications were set as carbamidomethyl (C) (fixed), deamidated (N) (variable), oxidation (M) (variable) and N-glycan modifications (182 in homo N-Glycan database; 309 in mammalian N-Glycan database). Up to two missed cleavage was allowed. The mass tolerance was set as ±5 ppm for the MS spectra and ±10 ppm for the MS/MS spectra. For glycopeptide identification, the Byonic score was over 100 and the false discovery rate (FDR) was less than 1%.

(24) Result

(25) This invention used α2-3,6,8 Neuraminidase to remove N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5GC) residues on the porcine fibronectin. The results showed that the porcine fibronectin after modification could be used in wound healing and play the same function as homo fibronectin (FIG. 3).

Example 3. Homo and Porcine Plasma Fibronectin Show Comparable Effects in Terms of Cell Adhesion and Migration

(26) Wound Healing Analysis

(27) U2OS, Hela or HFF1 cells growing on tissue culture plates were trypsinized and re-seeded on 10 μg/ml fibronectin-coated 6-well plates in the culture medium for 16 h and then placed in the temperature-controlled and CO2-controlled chamber of a microscope (Axio Observer.Z1, Zeiss) equipped with a 10×0.25 NA objective lens (Zeiss). Time-lapse images were obtained at 15-min intervals over 12 h using an AxioCamMR3 CCD camera operated by the Zen image analysis software (Zeiss). To calculate the percentage of wound closure, the wound area over a 6-h period or a 12-h period of migration was obtained from the time-lapse movies using the Metamorph image analysis software (Molecular Device), and calculated as the ratio of net wound-healing area to the wound area at 0-h after wounding.

(28) Result

(29) To compare the functionalities of the isolated fibronectin from homo and porcine plasma, we carried out wound-healing migration assays using cells that had been plated on the fibronectin-coated plates. This revealed that the isolated homo and porcine fibronectin proteins exhibited similar wound closure effect using U2OS, HFF1 and Hela cells (FIG. 4a-c). Therefore, homo and porcine fibronectins would seem to possess comparable capabilities in terms of regulating cell migration.

(30) Adhesion Assay

(31) The cell adhesion assays used 96-well plates that had been pretreated with 1% denatured BSA at 37° C. for 1 h and then coated with the indicated concentration of homo or porcine plasma fibronectin. To perform the experiments, U2OS cells growing on tissue culture plates were trypsinized, re-suspended in serum-free medium and then re-seeded on the pre-treated 96-well plates for 10 min or overnight (˜16 h). After incubation, any non-attached cells were removed completely by washing with PBS twice, and adherent cells were fixed with 5% glutaldehyde in H.sub.2O for 25 min at room temperature, flowed by staining with 0.1% crystal violet in H.sub.2O for 25 min at room temperature. After removing any un-bound crystal violet, the crystal violet-labelled adherent cells were solubilized in 50□ solution A (50% ethanol and 0.1% acetic acid in H.sub.2O), and the amount of crystal violet present measured using a Thermo Scientific Multiskan Spectrum at OD 550 nm. The results are presented graphically using Excel software (Microsoft).

(32) Immunofluorescence Staining and Image Analysis

(33) For paxillin staining, the cells were fixed and immunostained using a method previously described. For TIRFM imaging, the cells were mounted on slides with PBS containing N-propyl gallate. TIRFM images were obtained using 100×1.49NA (Oil-Immersion) Plan objective lens (Nikon) using the iLas multi-modal of the TIRF (Roper)/spinning disk confocal (CSUX1, Yokogawa) microscope system equipped with an Evolve EMCCD camera (Photometrics). To determine the adhesion area, TIRFM images of paxillin-stained cells were thresholded to highlight only the FAs and the areas of these regions were recorded using Metamorph. The total area of recorded FAs was summed to give the adhesion area. The results are presented graphically using Excel software (Microsoft).

(34) Cell Spreading Assay and Image Analysis

(35) Cells growing on tissue culture plates were trypsinized and re-seeded on plates coated with the indicated concentration of homo or porcine plasma fibronectin to allow them to adhere and spread (30 min). Next the cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and then imaged using a microscope 22 (Eclipse TS100; Nikon) coupled with a 20×0.45NA objective lens (Nikon) and a WHITE CCD camera operated by ISCapture software (TUCSEN). To calculate the cell spreading area, the cell area was manually circled on the phase images using Metamorph image analysis software (Molecular Device) and the results are presented graphically using Excel software (Microsoft).

(36) Result

(37) To determine whether homo and porcine fibronectins are comparable when regulating adhesion strength, we initially compared the effect of homo and porcine fibronectin on cell spreading and adhesion. The area of cell spreading was measured after 30 min using U2OS cells that had been seeded onto plates coated with increasing concentrations of homo or porcine fibronectin (FIG. 5). The results revealed that the area of cell spreading increased as the concentration of fibronectin increased for both fibronectins (FIG. 6). Cell adhesive capacity was also quantified and this revealed that increasing concentrations of homo or porcine fibronectin promoted cell adherence to fibronectin (FIG. 7). Next, we immunolabelled and visualized the cellular pattern of the FA marker paxillin (FIG. 8) using cells seeded on increasing concentration of homo or porcine fibronectin for 1.5 h. The results showed an increased area of adhesion (μm.sup.2) as the concentration of each fibronectin increased; the quantification was in terms of the area of paxillin-marked adhesion (FIG. 9).

(38) To further determine whether sialic acids (Neu5Ac and Neu5GC) on fibronectin do not responsible for the functioning of fibronectin during adhesion enhancement, we first used α2-3,6,8 Neuraminidase (sialidase) to cleave sialic acids from porcine fibronectin (FIG. 3). We found that the removal of sialic acids from porcine fibronectin did not affect the cell-fibronectin association (FIG. 10). Therefore, the sialic acid-trimming glycosylated fibronectin can be used in for novel wound dressing materials in clinical application to enhance wound healing without the possibility of an aberrant immune response caused by the presence of Neu5Gc.

Example 4. Porcine Plasma Fibronectin Shows Better Effects in Wound Closure after Proteinase Digestion

(39) MMP3 Digestion of Porcine Plasma Fibronectin

(40) To have better exposure the glycan structures on porcine fibronectin for better wound closure function. This invent provide a method for modify the glycosylated porcine fibronectin, comprising: incubating porcine plasma fibronectin with MMP3 overnight at 37° C. at an enzyme-substrate ratio from 1:5 to 1:30.

(41) Wound Healing Analysis

(42) U2OS cells growing on tissue culture plates were trypsinized and re-seeded on 10 μg/ml fibronectin or digested fibronectin-coated 6-well plates in the culture medium for 16 h and then placed in the temperature-controlled and CO2-controlled chamber of a microscope (Axio Observer.Z1, Zeiss) equipped with a 10×0.25 NA objective lens (Zeiss). Time-lapse images were obtained at 15-min intervals over 12 h using an AxioCamMR3 CCD camera operated by the Zen image analysis software (Zeiss). To calculate the percentage of wound closure, the wound area over a 12-h period of migration was obtained from the time-lapse movies using the Metamorph image analysis software (Molecular Device), and calculated as the ratio of net wound-healing area to the wound area at 0-h after wounding.

(43) Result

(44) This invention used MMP3 to generate porcine fibronectin peptides (FIG. 11). To compare the effect of digested fibronectin peptides and non-digested fibronectin, we carried out wound-healing migration assays using cells that had been plated on the digested fibronectin peptides- or non-digested fibronectin-coated plates. This revealed that digested fibronectin peptides significantly promote wound closure effect (FIG. 12). Therefore, the fibronectin peptides possess enhanced ability in terms of regulating cell migration.