FUNCTIONALIZING NANOFIBRES

Abstract

Methods for functionalizing the surface of nanofiber substrates, including electrospun fibres and non-woven or woven mats of fibres are described. Functionalised nanofiber substrates presenting biologically active moieties such as biotin and saccharides are described.

Claims

1-15. (canceled)

16. A functionalized nanofibre comprising an electrospun admixture of a polymer and a water soluble synthetic construct of the structure F-S-L where F is a functional moiety, L is a lipid and S is a spacer linking F to L via covalent bonds.

17. The functionalized nanofibre of claim 16 where the polymer is cellulose acetate (CA) or poly[(2-propyl-1,3-dioxane-4,6-diyl)methylene] (PVB).

18. The functionalized nanofibre of claim 17 where F is a functional moiety selected from the group consisting of: fluorophores of fluorescein, biotin, monosaccharides, disaccharides, trisaccharides and oligosaccharides.

19. The functionalized nanofibre of claim 18 where L is a phosphatidyl-ethanolamine.

20. The functionalized nanofibre of claim 19 where S is selected from the group consisting of: ##STR00005## where m is the integer 1, 2 or 4, n is the integer 3, 4 or 5, p is the integer 1, 2 or 3 and q is the integer 2, 3, 4, 5 or 6.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1. A schematic representation of the apparatus used to prepare the electrospun nanofibres.

[0028] FIG. 2. Photomicrographs of nanofibres electrospun from a dispersion of PVB and the construct designated KODE™-fluorescein (I) before (A and B) and after (B and D) washing with methanol tipper photomicrographs (A and B) are at higher magnification.

[0029] FIG. 3. Photographs of immunostained samples of mats of nanofibres electrospun from dispersions of PVB alone (A), PVB in admixture with the construct designated KODE™-biotin (II), PVB in admixture with biotin par se (C), and PVB in admixture with the construct designated KODE™-biotin (II) after washing with methanol prior to immunostaining (D).

[0030] FIG. 4. Photographs of immunostained samples of mats of nanofibres electrospun from dispersions of PVB alone (A), PVB in admixture with the construct designated KODE™-A.sub.tri (III) (B), and PVB in admixture with the construct designated KODE™-A.sub.tri (III) with a methanol wash prior to being immunostained (C).

[0031] FIG. 5. Photograph of immunostained samples of mats of nanofibres electrospun from a dispersion of PVB alone or in admixture with one of the constructs designated KODE™-biotin (II) (‘FSL-biotin’), KODE™-A.sub.tri (III) (‘FSL-A’) or biotin per se. Immunostaining was performed with anti-A antibody.

[0032] FIG. 6. Scanning electron micrographs of nanofibres electrospun from dispersions of PVB alone (A and B), PVB in admixture with the construct designated KODE™-biotin (II) (C and D), PVB in admixture with the construct designated KODE™-fluorescein (I) (E and F) and PVB in admixture with the construct: designated KODE™-A.sub.tri (III) (G and H). Micrographs B, D, F and H are at higher magnification.

[0033] FIG. 7. Immunostaining with alkaline phosphatase conjugated streptavidin of the surface of non-woven mats of nanofibre (5GSM nylon (Upper), 1GSM nylon (middle) and 3GSM PCL (lower)) printed with an aqueous dispersion of either the construct designated KODE™-A.sub.tri (III) or the construct designated KODE™-biotin (II) (B). The identity of the substrate and dispersion employed is identified by the words appearing following immunostaining. All substrates sourced from Revolution Fibres (Henderson, Auckland, New Zealand).

[0034] FIG. 8. Immunostaining with alkaline phosphatase conjugated streptavidin of the surface of nanofibre mats (5GSM nylon (Upper), 1GSM nylon (middle) and 3GSM PCL (lower)) printed with a dispersion of the construct designated KODE™-A.sub.tri (III) using polyclonal serum.

[0035] FIG. 9. Immunostaining with alkaline phosphatase conjugated streptavidin of the surface of nanofibre mats (5GSM nylon (Upper), 1GSM nylon (middle) and 3GSM PCL (lower)) both before (first and third rows) and after (second and fourth rows) washing with methanol.

[0036] FIG. 10. Photograph of red blood cells modified to incorporate the construct designated KODE™-biotin (II) (kodecytes) attached to avidinylated (streptavidin) substrate following printing with the construct designated KODE™-biotin (II).

[0037] FIG. 11. Scanning electronmicrograph images of red blood cells attached to discrete areas of avidinylated (streptavidin) nanofibre mats following printing with the construct designated KODE™-biotin (II).

DETAILED DESCRIPTION

[0038] The biologically active moieties are introduced in the form of water dispersible lipid conjugates (constructs) of the generic structure F-S-L where F is the functional moiety, L is the lipid and S is a spacer covalently linking F to L. Without wishing to be bound by theory it is believed that the amphipathic nature of the constructs favours expression of the functional moiety at the surface whether the moiety is introduced at the time the fibres are electrospun or following formation of a mat of electrospun fibres. In the latter circumstance the constructs may be printed onto the surface of the mat of electrospun fibres in the form of an aqueous dispersion using conventional ink let printing techniques. Higher resolution is achievable when the constructs are printed onto a nanofibre substrate, such as a mat of electrospun fibres. In either case, the functionalization of the surface of the electrospun nanofibres is achieved with greater efficiency then would be achieved by preparing a dispersion of polymer and functional moiety alone.

[0039] Constructs of the structure F-S-L that may be added to dispersions of these polymers include the constructs designated KODE™-fluorescein (I), KODE™-biotin (II) and KODE™-A.sub.tri (III):

##STR00002## [0040] I (where F is fluorescein thiocyanate);

##STR00003## [0041] II (where F is biotin); and

##STR00004## [0042] III (where F is A.sub.tri).

[0043] The preparation of the construct designated KODE™-fluorescein (I) is described in the publication of Korchagina et al (2008). The preparation of the construct designated KODE™-biotin (II) is described in the publication of Bevin et al (2009). The preparation of the construct designated KODE™-A.sub.tri (III) is described in the publication of Bovin et al (2005).

[0044] Experimental

[0045] Preparation of Electrospun Nanofibres

[0046] Samples of functionalized nanofibres were prepared using the apparatus illustrated schematically in FIG. 1. Briefly, a dispersion of polymer is dispensed into an elongated reservoir (1). A star shaped metal wheel (2) rotates in the direction indicated in FIG. 1 and picks up droplets of the dispersion on the points (3) of the wheel (2). A high voltage is maintained between the wheel (2) and the metal collector plate (4) to which a collecting surface such as a cloth or paper backing is affixed. As the wheel (2) rotates the electric field strength overcomes the surface tension of the dispersion and a charged polymer jet is ejected from the pendant droplets carried on the points of the wheel (3) towards the plate (4) causing a mat of fibres (5) of the polymer to be deposited on the backing affixed to the plate (4). Dispersions of PVB when, prepared at a concentration of 10% (w/v) in 100% ethanol. Dispersions of CA were prepared at a concentration of 17% (w/v) in 70:15:15 (v/v/v) MEK/DMA/DMF. Mats of nanofibres electrospun from these dispersions with or without the addition of KODE™ constructs or biotin were used in the following experiments.

[0047] Speed of rotation, the distance to the collector plate and voltage were readily adjusted to optimise deposition of the nanofibres electrospun from each of the dispersions. Where KODE™ constructs were added to the dispersions 50 μL of a 10 mg/mL solution of the construct in 100% ethanol was added to a 5 mL volume of the dispersion of the polymer to provide a final concentration of 130 μg/mL. It is anticipated that the deposition of nanofibres can be further optimised by adjustment of the polymer concentration, medium volatility and medium conductivity. Where biotin was added to the dispersions 500 μL of a 250 μg/mL concentration of the solute in 100% ethanol was added to a 5 volume of the dispersions of the polymer to provide a final concentration of 25 μg/mL.

[0048] Immunostaining of Electrospun Nanofibres

[0049] Mats of nanofibres electrospun from the dispersions were immunostained on their backing papers following blocking of the surface with 2% BSA for 60 minutes. For mats of nanofibres electrospun from dispersions containing the construct designated KODE™-biotin (II) and the respective controls, a 1 μg/mL solution of streptavidin conjugated to alkaline phosphatase (s2890, Sigma) in 2% (w/v) BSA was incubated on the surface at room temperature for 30 minutes. The surface was then washed 6 times in PBS before incubating at room temperature with a 50-fold dilution of NBT/BCIP substrate (11 681 451 001, Roche) in 2:2:1 (mol/mol/mol) Tris-NaCl—MgCl2 buffer (pH 9.5) for, about 2 minutes. The samples were then washed with PBS and air dried.

[0050] For mats of nanofibres electrospun from dispersions containing the KODE™ construct designated KODE™-A (III) and the respective controls, a 5-fold dilution of a mouse anti-A antibody (epiclone, CSL) in 2% BSA was incubated on the surface at room temperature for 30 minutes. The surface was then washed 6 times in PBS before incubating at room temperature with a 1000-fold dilution of alkaline phosphatase conjugated anti-mouse antibody (AQ502A, Millipore) in 2% BSA for 30 minutes. The surface was washed again (6 times, PBS) before incubating with the chromogenic substrate as before. Photomicrographs and photographs of the mats of functionalized nanofibres following immunostaining and their respective controls are provided in FIGS. 2 to 5. Scanning electronmicrographs of the electrospun fibres are provided in FIG. 6.

[0051] Printing on Nanofibres

[0052] The ability of mats of nanofibres to serve as an improved substrate for the printing of FSL constructs as described in the publication of Bovin et al (2011) was evaluated. Solutions of the construct designated KODE™A.sub.tri (III) (FSL-A) and the construct designated KODE™-biotin (II) (FSL-Biotin) were printed on to mats of nanofibres (Revolution Fibres, Henderson, Auckland, New Zealand). An ink jet printer (EPSON STYLUS™ T21) with refillable cartridges modified to hold a smaller volume was employed. The constructs were prepared as solutions and used to fill separate modified cartridges. To facilitate identification and as an illustration of one of the advantages provided by printing FSL constructs the identification of the source of the mats, type of nanofibre and solution were printed. Following printing of the solutions each sample was blocked and immunostained as before. The immunostained samples are presented in FIGS. 7 to 9.

[0053] Immobilisation of Cells

[0054] The ability of the printing technique to immobilise red blood cells modified to incorporate the construct designated KODE™-biotin (II) (kodecytes) to discrete areas following avidinylation (streptavidin) of mats of nanofibres also printed with the construct is shown in FIGS. 10 and 11.

[0055] Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specific elements, features or integers, such equivalents are incorporated as if specifically referred to in this specification. In particular, variations and modifications to the embodiments or examples that include elements, features or integers disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description may be provided in the alternative or in combination in these different embodiments of the invention.

REFERENCED PUBLICATIONS

[0056] Bovin et al (2005) Synthetic membrane anchors international application no. PCT/NZ2005/000052 (publ. no. WO 2005/090368 A1)

[0057] Bovin et al (2009) Functional lipid constructs international application no. PCT/NZ2008/000266 (publ. no. WO 2009/0483434 A1)

[0058] Bovin et al (2011) Printing of constructs international application no. PCT/NZ2010/000127 (publ. no. WO 2011/002310 A1)

[0059] Garca et al (2013) Nonwoven membrane as a drug delivery system International application no. PCT/EP2013/056522 (publ. no. NO 2013/144206 A1)

[0060] Kiick et al (2006) Multifunctional and biologically active matrices from multicomponent polymeric solutions U.S. patent application Ser. No. 11/395,699 (publ. no. US 2006/0240313 A1)

[0061] Korchagina et al (2008) Fluorescent cell markers International application no. PCT/NZ2007/000256 (publ. no. WO 2008/030115 A2)

[0062] Seiler et al (2009) Nanofibre matrices formed from electrospun hyperbranched polymers International application no. PCT/EP2009/055045 (publ. no. WO 2009/133059 A2)

[0063] Supaphol et al (2012) Electrospinning of biocompatible polymers and their potentials in biomedical applications Advances in Polymer Science, 246, 213-239

[0064] Tojo et al (2013) Nanofibre U.S. patent application Ser. No. 13/703,315 (publ. no. US 2013/0125912)

[0065] Vile et al (2013) Bioactive nanofibres International application no. PCT/IB2012/054626 (publ. no. WO 2013/035072 A1)