POLYSACCHARIDE NANOFIBRES HAVING ANTIMICROBIAL

PROPERTIES

The present invention relates to polysaccharide nanofibres having anti microbial properties and a method of making them. In particular the invention relates to polysaccharide nanofibres having silver nanoparticles dispersed throughout the fibres. The fibres may be produced by electrospinning and may be used in wound care.

The incorporation of silver into fibrous wound dressings is known. Generally the silver is held on the surface of the fibres or dressing. Although this imparts anti microbial properties to the dressing, it can lead to several disadvantages. Excess silver may need to be used because, due to its presence at the surface, the silver may be released or made inactive quickly. The excess, while providing a reservoir can result an unacceptable physical appearance of the dressing due to discolouration of the silver or may result in staining of the skin of the patient. The incorporation of particles into fibrous wound dressings has been described in US 7229689 but the method of incorporation involves the addition of silver from an ion exchange resin in order to avoid discolouration. It would be desirable to use silver in a fibrous dressing in such a manner that the silver is distributed evenly through the fibres so that a sustained release of silver is obtained from the dressing. It would also be desirable to use silver in the form of nanoparticles as silver nanoparticles have been shown to possess antimicrobial properties and present a larger surface area for release.

WO 2005/073289 discloses the mixing of metal particles with a polymer dope, prior to extrusion and solidification into fibres or films. One of the problems associated with the incorporation of nanoparticles into fibres is the difficulty of dispersing the particles uniformly as particles tend to agglomerate.

Electrospinning is a well known fabrication technique, which can be used to produce polymer fibres in the range lnm to lμm. The process of electrospinning polymer solutions involves the formation of an electrically charged liquid jet from the surface of a polymer solution in the presence of an electric field. The liquid jet undergoes stretching effects and drying as the solvent evaporates, and is deposited as polymer fibre on a suitably positioned, oppositely charged target. These electrospun polymer nanofibres are most commonly deposited in the form of a non- woven web.

In the past, relatively few natural polymers were successfully electrospun into nanofibres. Whereas synthetic polymers can have carefully controlled molecular weight and molecular weight distribution and are typically produced with long, flexible, linear chains, natural polymers are generally more complex and have strong hydrogen bonding, which leads to relatively low chain flexibility. This often results in natural polymers with unfavourable conformations.

We have found that it is possible to produce polysaccharide nanofibres with anti microbial properties. In particular we have found that it is possible to incorporate silver particles into polysaccharide nanofibres.

Accordingly, a first aspect of the present invention provides polysaccharide nanofibres having anti-microbial properties said nanofibres comprising alginate and having silver nanoparticles dispersed throughout the fibres.

Such fibres have the advantage that they present a large surface area for delivery of silver to a wound. They may also have the advantage that the silver is released to the wound in a sustained manner. By the term dispersed throughout the fibre is meant that the nanoparticles are distributed within the fibres. The particles may be distributed through the whole thickness of the fibre and preferably are uniformly distributed. In this way a predictable dosage of silver may be delivered to the wound.

By the term nanoparticle is meant a particle having a diameter of from lnm to lOOnm, generally between l-50nm and preferably between 1-lOnm.

By the term nanofibre is meant a fibre having a diameter of less than 1 micron, generally between 1 and 500nm, preferably between 20-500nm.

Preferably the silver particles are present in the fibres at a concentration of between 0.002% (w/w) and 2% (w/w), more preferably between 0.02% (w/w) and 1% (w/w).

The polysaccharide nanofibres are preferably gel forming fibres by which is meant that the fibres are hygroscopic fibres which upon the uptake of wound exudate become moist, slippery or gelatinous and thus reduce the tendency for the surrounding fibres to adhere to the wound. The gel forming fibres can be of the type which retain their structural integrity on absorption of exudate or can be of the type which lose their fibrous form and become a structureless gel. The gel forming fibres may comprise in addition to alginate, sodium carboxymethylcellulose, pectin, chitosan, hyaluronic acid, or other polysaccharides. The gel forming fibres preferably have an absorbency of at least 2 grams of 0.9% saline solution per gram of fibre (as measured by the free swell method) . Preferably the gel forming fibres have an absorbency of at least 10g/g as measured in the free swell absorbency method, more preferably between 15g/g and 25g/g.

Alginate is a natural polysaccharide existing widely in many species of brown seaweeds. The alginate for use in the present invention can be sodium alginate of the type containing a high proportion of guluronate but can also be of the type containing a high proportion of mannuronate.

The polysaccharide nanofibres may be produced by electrospinning. We have found that polysaccharide nanofibres produced by electrospinning advantageously may have silver nanoparticles uniformly dispersed throughout the fibres. The distribution can be measured by transmission electron microscopy.

A second aspect of the invention relates to an aqueous solution for spinning polysaccharide nanofibres, said solution comprising:

from 2% (w/w) to 8% (w/w) of sodium alginate from 0.05% (w/w) to 5% (w/w) of water soluble polymer and from 0.00015% (w/w) to 0.2% (w/w) of silver compound.

Preferably the solution contains from 0.1% by weight to 1% by weight of a water soluble polymer such as polyethylene oxide, polyvinyl alcohol or polyvinyl pyrrolidone or a mixture thereof. More preferably the water soluble polymer has a long-chain linear structure and high molecular weight.

The solution may also comprise from 2% by weight to 20% by weight of a polar aprotic solvent such as DMSO to break down hydrogen bonding within the polysaccharide and improve the polymer chain entanglement during electrospinning. The solution may also comprise from 0.01 % w/w to 1% w/w of non-ionic surfactant such as Triton X-100 to alter the surface tension of the solution.

Preferably, the aqueous solution of sodium alginate has a weight proportion of PEO to alginate ratio between 2% and 25% and a DMSO concentration between 5% (w/w) and 10% (w/w), with small concentrations of silver nitrate. Advantageously silver nanoparticles can be formed in-situ in such a solution by photochemical reduction of a silver compound such as silver nitrate. Silver nanoparticles are formed when silver ions dissociate from a silver compound when it is dissolved, and gain an electron in an oxidation-reduction reaction with

a reducing agent such as carboxyl and/or hydroxyl groups of polymers. This results in silver atoms which act as seeds onto which other silver ions are reduced, resulting in clusters of silver atoms which grow into nanoparticles as more silver accumulates and clusters join together. These solutions can then be electrospun to form nanofibres with diameters in the range lnm - lμm, which desirably contain a uniform distribution of silver nanoparticles.

Accordingly a third aspect of the invention relates to a process for forming polysaccharide nanofibres by:

a) making a solution comprising

- from 2% (w/w) to 8% (w/w) of sodium alginate from 0.05% (w/w) to 5% (w/w) of water soluble polymer and from 0.00015% (w/w) to 0.2% (w/w) of silver compound and

b) electrospinning the solution to form nanofibres.

The electrospun nanofibres may then be ionically cross-linked in a bath containing excess calcium ions, in order to transform some or all of the sodium alginate to calcium alginate. The calcium alginate or sodium/calcium alginate nanofibres, containing silver nanoparticles may then be soaked in water the remove the excess calcium, before being dried. Preferably the dried fibres comprise calcium alginate and sodium alginate in the ratio of 80% calcium alginate to 20% sodium alginate.

Preferably the solution is prepared in ambient light and then stored in the dark prior to electrospinning within 12 hours of preparation, more preferably within 6 hours of preparation and more preferably within 4 hours of preparation.

Preferably the solution has a viscosity prior to spinning of between lPa:s and 10 Pa:s.

More preferably the solution comprises an anti-agglomeration agent such as a non-ionic triblock copolymer or an organoalkoxysilane.

The invention is illustrated by the following figures in which:

Figure 1 a) shows a UV-visible spectra showing the development of silver particles in alginate solution containing 5mmol.L ' AgNO ₃ ; b) growth of the 450nm peak for alginate solutions containing a range of AgNO ₃ concentrations both in ambient light conditions;

Figure 2 shows TEM images of electrospun alginate nanofibres containing silver particles electrospun a) after 7 days; and b) within 4 hours of preparation (micron bars: 200μm). Image c) shows a higher magnification image of sample b) (micron bar: 500μm);

Figure 3 shows EDX spectrum of a silver nanoparticle within the alginate fibres; and

Figure 4 shows electrospun alginate discs, on nutrient agar plates covered by a lawn of s. aureus, a) without silver; and c) containing silver nanoparticles. b) and d) are close-ups of samples from a) and c) respectively.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLE 1

PEO (Mw: > 5 OOOOOOg.mol ') was dissolved in deionised water to a concentration of 1-4 % (w/w). The solution was stirred until it appeared homogenous. After allowing time for degassing, a calculated mass of the PEO solution was mixed into a known mass of a solvent consisting of DMSO and deionised water, with a DMSO concentration between 2% (w/w) and 20%

(w/w), preferably between 5% (w/w) and 10% (w/w). Sodium alginate was then slowly added to a vortex in the PEO/water/DMSO solution such that the total polymer concentration in the solution was between 3% and 8% (w/w), preferably between 5% and 6% (w/w) and the PEO to Alginate ratio was between 2% and 10% by weight, preferably between 2% and 5% by weight. The solution was stirred thoroughly until it was consistently viscous and homogenous. Additions of the surfactant Triton X-IOO were made, using a micropipette to a vortex in the alginate solution, such that the concentration was varied between 0.1% and 1% (w/w) .

In another solution, the deionised water was partially or entirely substituted for a dilute solution of AgNO ₃ , before the alginate was added, such that the AgNO ₃ concentration in the alginate solution was between O.lmmol.L and 10 mmol.L ^"1 .

In another solution, a known volume of a 0.1 mol.L ' aqueous solution of AgNO ₃ was added to the alginate using a micropipette, such that the final concentration of AgNO) in the alginate solution was between 0.1 and 10 mmol.L '.

In another solution, PEO (Mw 600,000-1,100,000 g.mol ') was used instead of PEO (Mw: > 5 OOOOOOg.mol ¹ ). In this solution the proportion of PEO to alginate ratio used was in the range 10% to 40% by weight, preferably 15% to 25% by weight.

These solutions were either centrifuged for 3 to lOmins at 2000rpm to 4000 rpm to remove air bubbles from the solution, or they were simply left until the solutions were clear of bubbles.

It was found that as soon as silver nitrate was mixed into the polymer solution in ambient light conditions, a reduction reaction took place. This caused a colour change in the solution, from the clear yellow of an alginate solution to a dark pink or grey over time. The results of spectrophotometry confirmed these

observations and can be seen in Figure 1. It can be seen that over the first four hours after preparation of the silver containing solutions, the absorbance increases rapidly. From then on the rate of increase is reduced. The development of multiple peaks and a broadening of the peak in Figure Ib indicate that as time progresses the silver particles grow and become aggregated.

The effect of solution aging time, that is to say the time between preparation and electrospinning, on the morphology and distribution of silver particles in the alginate fibres can clearly be seen in Figure 2. The sample produced 7 days after solution preparation has large aggregated silver particles, non-uniformly distributed, whereas the sample electrospun from fresh solution contains more evenly distributed silver particles, which are significantly smaller. With shorter solution aging times the aggregation of silver nanoparticles is reduced. It has also been found that if solutions are stored in the dark after an initial one hour aging time, particle growth and aggregation is inhibited so that alginate fibres with uniformly distributed silver nanoparticles can more easily be produced.

The alginate solutions were electrospun from a stainless steel needle of gauge size between 22 G and 31 G, which was connected to a syringe. Solution was maintained at the tip of the needle by means of a digitally controlled syringe pump, such that the flow rate was in the range 10-30 μl.min '. An applied voltage in the range 5kV to 30 kV, preferably 1OkV to 20 kV was applied to the needle, which was positioned between 10cm and 50 cm, preferably between 15cm and 25 cm away from the collector.

After electrospinning, nanofibrous webs were removed from the collector and ionically cross-linked in a bath either containing an aqueous solution of CaCl ₃ , an organic solution of CaCl ₃ followed by an aqueous solution of CaCl ₃ , or an aqueous organic solution of CaCl _3. After cross-linking the fibres were soaked in either deionised water, or a mix of water and organic solvent, in order to remove

any excess CaCl ₃ Or resulting NaCl from the fibres. Samples were then dried before characterisation.

The electrospun alginate samples were characterised using scanning electron microscopy (SEM) , transition electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX). Samples, taken for SEM before and after cross- linking were mounted on aluminium stubs and sputter coated with IOnm Pt/Pd before imaging. TEM samples were collected on carbon coated copper grids during electrospinning.

In order to test for antimicrobial efficacy, samples of the cross-linked alginate fibres with and without silver nanoparticles were punched into 8mm diameter disks and sterilised in 100% ethanol before use.

Staphylococcus aureus, a common wound pathogen, was grown in nutrient broth overnight and then used to inoculate nutrient agar plates, to create a lawn of bacteria. The sample discs were then placed onto the agar plates and incubated at 37 ⁰ C for approximately 15 hrs. In this time the lawn of s. aureus grew to form visible colonies on the agar plates. Inhibition of the growth of these colonies around the sample discs is an indicator as to the antimicrobial efficacy of the material.

Results of the antimicrobial sensitivity assay can be seen in Figure 4. It is clear that the electrospun alginate samples have no inhibitory effect on the growth of the s. aureus colonies, whereas the samples containing silver nanoparticles all inhibited the growth of the bacterial colonies directly under the discs as well as in zone around the discs.

The electrospun webs were also characterised for release into water and Solution A. Solution A is an aqueous solution with physiological concentrations of sodium chloride and calcium chloride. The release rate was found to reduce after

three or four days of immersion in Solution A although even after two weeks, silver was being released. This demonstrates the desirable sustained release of silver from electrospun alginate webs.

EXAMPLE 2

The second example describes the addition of a stabilising agent in the process described above, which restricts the growth of the silver nanoparticles and prevents them from aggregating. This allows nanofibres to be electrospun over a range of time periods, without losing the uniform distribution of fine silver nanoparticles.

The stabilising agent used is an aqueous amphiphilic tri-block copolymer consisting poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) blocks. This copolymer is capable of forming micelles around metallic nanoparticles, stabilising them as a colloid in the aqueous solution.