GB2532408A - Glycobiological surfaces - Google Patents

Abstract

A method for depositing a sugar molecule on a surface, said method comprising exposing a surface to a plasma containing a sugar monomer, under conditions in which the monomer is deposited on the surface so as to form a polysaccharide layer thereon. The sugar monomer may be mono-, di- or oligosaccharides or a monosaccharide linked to a polymerisable group such as a (meth)acrylate, alkenyl or alkynyl group. A substrate may be a polymer, glass or metal. A substrate with a plasma polymerised polysaccharide may be used as glycobiological sensor, model system or as biocompatible layer in implants. An example of a glycobiological sensor uses a nitrocellulose membrane or silicon nitride on an atomic force microscopy (AFM) tip, which is placed in a vacuum chamber; then β-D-galactose ethylmethacrylate (galEMA) and an inert gas are introduced; the plasma deposition and polymerisation happens under pulsed RF plasma conditions. Inert gases may be argon or preferably helium.

Description

Glycobiological Surfaces The present invention relates to methods for depositing sugar molecules onto surfaces, such surfaces having utility in medical, diagnostic or analytical devices, as well as surfaces and devices formed thereby.

Sugars perform highly specialised roles in many biological and biochemical processes and procedures. For example, sugars are important in many cell-cell interactions and protein folding which are governed by interactions between the binding proteins and the carbohydrate moieties present on the surface of the cells. The structure and orientation of the sugar molecules is important for these interactions. In many applications, they are immobilised on surfaces so as to produce a useful device, in particular an assay or microarray, or even a biocompatible surface on a medical device, such as an implant, stent or the like. They are used in forming model systems and sensors in glycobiology and as anti-adhesion agents in potential tumour treatments. For example by creating surfaces containing these chemistries on devices such as atomic force microscopy tips, when scanned across a biological surface a profile can be built up depending on the resultinc interactions. Also due to the similarity to chemistries within the body, a degree of bio-compatibility can be displayed.

In all cases, there is a need to modify a surface so that it carries sugar molecules in such a way that they are able to interact with a biomolecule, either a specific biomolecule or a cell, for example so that it becomes immobilised on the surface.

Currently surfaces with sugar molecules thereon are prepared using custom made chemicals, in solution based applications. 35 This limits the type of surfaces they can be applied to due to unfavourable solution energetics preventing certain chemicals being bonded to certain substrate media.

In these procedures, in essence, solutions of the sugars are applied to the surface and the solvent removed. The sugar then becomes associated with the surface material, however may not be well adhered.

The nature of the surface material will depend upon the nature of the device or filtration media being prepared. Where the device is for example a microtitre or assay plate, microarray slides, filtration component, implantable device or culture plates or the like, it is generally a polymeric material such as polystyrene, polyurethane, polyacrylates such as polymethacrylate, or polyalkylene such as polyethylene, polycarbonate or polypropylene, however could be made of any material including metal. Slides and culture plates may also be glass. Filtration and binding media may take various forms such as membranes, for example cellulose or nitrocellulose membranes, or binding media such as polystyrene, silica or magnetic beads. Generally however, functionalised surfaces are required for these items to operate efficiently.

In some cases, the surface may be pre-treated in a plasma chamber, either under vacuum or in the presence of a gas such as oxygen, so as to activate the surface and make it more receptive to the organic compound.

Surfaces modified using these techniques are not always as effective or efficient as they might be. Precise control over the density, packing and orientation of the sugar molecules is not possible. However, these factors can have an impact on the efficacy of the surface, and can make the difference between a successful or a failed experiment, or the efficiacy of the device or therapy.

Furthermore, the sugars are effectively arranged in a monolayer on the surface, potentially limiting the amount of sugars which can be attached to the surface.

According to the present invention there is provided a method for depositing a sugar molecule on a surface, said method comprising exposing a surface to a plasma containing a sugar monomer, under conditions in which the monomer is deposited on the surface so as to form a polysaccharide layer thereon.

As used herein, the expression "sugar" refers to any saccharide molecule, in particular monosaccharides, disaccharides and oligosaccharides, but in particular monosaccharides, such as glucose, fructose, ribose, arabinose, xylose, lyxose, allose, altrose, gulose, mannose, idose, galactose or talose as well as derivatives thereof. The derivatives may be any hybrid species which is required for the desired glycobiological function, but one such example is p-D-galactose ethylmethacrylate (GalEMA).

By utilising plasma deposition as a means of applying the sugar in the form of a polymer to the surface, close control may be maintained of the resultant polymer structure and therefore the density and availability of the sugar molecules. The deposition mode can be controlled and varied for instance by controlling the power of the plasma, whether or not the plasma is continuous or pulsed, the deposition time, the concentration of the monomer etc. By controlling these factors, the degree of fragmentation and rate and nature of deposition of the sugar can be selected, and thus the structure of the polymer formed on the surface can be controlled. The structure may even be varied as the polymer is grown, ensuring that the orientation of the sugar molecule is optimised for the desired purpose.

In this way, a polymeric layer including multiple sugar molecules at a predetermined density and orientation may be achieved.

Using this method, plasmas are generated from sugar molecules, which are subjected to an electrical field. When this is done in the presence of a substrate, the radicals of the sugar in the plasma polymerise on the substrate. Conventional polymer synthesis tends to produce structures containing repeat units that bear a strong resemblance to the monomer species, whereas a polymer network generated using a plasma can be extremely complex. The properties of the resultant coating can depend upon the nature of the substrate as well as the nature of the sugar used and conditions under which it is deposited.

The nature of the polymer coating applied in any particular case, will be determined by the desired end use. For instance, the nature of the sugar used will depend upon the type of saccharide groups required to be present in the final coating and the amount of such groups which are required to allow the surface to fulfil the function.

In some cases, it may be suitable to use a mixture of sugars so as to produce a surface which includes polymers which contain the same or different sugar molecules. The arrangement of the sugars within the polymer may be designed to that they are favourably orientated in the final polymer layer.

In addition, further sugars may he arranged along the polymer 30 chain, ensuring effective capture or binding of the target biomolecule in three-dimensions.

Thus sugars which may be utilised in the process include for example sugar derivatives, for example saccharides, and in 35 particular monosaccharide molecules, which are covalently linked to polymerisable groups. Suitable polymerisable groups which include unsaturated groups such as acrylate, alkenyl or alkynyl groups. Any unsaturated groups or acylate groups in the derivatives will have the effect of favouring or steering the polymerisation and so the arrangement of these groups can be selected so as to achieve the desired orientation of the saccharide section itself, in the final polymer.

Acrylates may suitably comprise alkylacrylates such as Cl 2ualkylacrylates, and in particular Ci Ealkylacrylates, such as methacrylates or ethacrylate groups. Similarly, alkenyl or alkynyl groups may contain from 2-10 carbon atoms and preferably from 2-10 carbon atoms. Any alkyl, alkenyl or alkynyl groups may be straight chain or branched. These groups may contain substituents, for example comprising heteroatoms such as oxygen, nitrogen or sulphur, provided that these do not adversely affect the final functionality of the polysaccharide layer.

However, using the plasma technique, it is possible to 20 polymerise even saturated sucars or derivatives as the activation converts the monomer compounds into radicals which are able to combine together to form polymeric moieties.

However, where densely packed sugars may be required, high power conditions, leading to significant fragmentation and/or the use of small chain monomers may lead to a more suitable coating.

In particular, the sugars molecules used as monomers should be 30 sufficiently volatile to allow them to be introduced into a plasma chamber in a gas phase.

In the method, in general, the substrate to be treated is placed within a plasma chamber together with one or more 35 sugars, which are able to generate the target polymeric substance, in an essentially gaseous state, a glow discharge is ignited within the chamber and a suitable voltage is applied.

As used herein, the expression "in an essentially gaseous state" refers to gases or vapours, either alone or in mixture, as well as aerosols.

The gas present within the plasma chamber may comprise a vapour of the sugar alone, but it may be combined with a carrier gas, in particular, an inert gas such as helium or argon. In particular helium is a preferred carrier gas as this can minimise fragmentation of the sugar monomer.

When used as a mixture, the relative amount of the sugar vapour to carrier gas is suitably determined in accordance with procedures that are conventional in the art. The amount of sugar added will depend to some extent on the nature of the particular sugar being used, the nature of the substrate being treated, the size of the plasma chamber etc. Generally, in the case of conventional chambers, monomer is delivered in an amount of from 50-250mg/min, for example at a rate of from 100-150mg/min. Carrier gas such as helium is suitably administered at a constant rate for example at a rate of from 5-00, for example from 15-30sccm. In some instances, the ratio of sugar to carrier gas will be in the range of from 100:0 to 1:100, for instance in the range of from 10:0 to 1:100, and in particular about 1:0 to 1:10. The precise ratio selected will be so as to ensure that the flow rate required by the process is achieved.

The plasma used may be continuous wave or pulsed depending Using pulsed plasmas, in which low average powers can be achieved, a highly controllable surface covering can be obtained with minimal deterioration of the sugar monomer, which is particularly important when retention of the sugar structure in the target polymer is required.

The applied fields are suitably of power of from 20 to 500W, suitably at about 100W peak power. When applied as a pulsed field, the pulses are suitably applied in a sequence which yields very low average powers, for example in a sequence in which the ratio of the time on: time off is in the range of from 1:3 to 1:1500, depending upon the nature of the monomer gas employed. Although for monomers which may be difficult to polymerise, time on: time off ranges may be at the lower end of this range, for example from 1:3 to 1:5, many polmerisations can take place with a time on:time off range of 1:500 to 1:1500. Particular examples of such sequence are sequences where power is on for 20-50ps, for example about 30ps, and off for from 1000ps to 30000lis, in particular about 20000ps.

Typical average powers obtained in this way are 0.01W.

The fields are suitably applied from 30 seconds to 90 minutes, preferably from 5 to 60 minutes, depending upon the nature of the sugar and the substrate, and the nature of the target coating required.

Suitable plasmas for use in the method of the invention include non-equilibrium plasmas such as those generated by radiofrequencies (Rf), microwaves or direct current (DC). They may operate at atmospheric or sub-atmospheric pressures as are known in the art. In particular however, they are generated by radiofrequencies (Pi).

Various forms of equipment may be used to generate gaseous plasmas. Generally these comprise containers or plasma chambers in which plasmas may be generated. Particular examples of such equipment are described for instance in W02005/089961 and W002/28548, but many other conventional plasma generating apparatus are available.

In all cases, a glow discharge is suitably ignited by applying a high frequency voltage, for example at 13.56MHz. This is applied using electrodes, which may be internal or external to the chamber, but in the case of larger chambers are internal.

Suitably the gas, vapour or gas mixture is supplied at a rate 5 of at least 1 standard cubic centimetre per minute (sccm) and preferably in the range of from 1 to 100sccm.

In the case of the sugar vapour, this is suitably supplied at a rate of from 80-300mg/minute, for example at about 120mg per 10 minute depending upon the nature of the sugar, whilst the pulsed voltage is applied.

Gases or vapours may be drawn or pumped into the plasma region. In particular, where a plasma chamber is used, gases or vapours may be drawn into the chamber as a result of a reduction in the pressure within the chamber, caused by use of an evacuating pump, or they may be pumped, sprayed, dripped, electrostatically ionised or injected into the chamber as is common in liquid handling.

Polymerisation is suitably effected using vapours of sugar monomers which are maintained at pressures of from 0.1 to 400mtorr, suitably at about 10-100mtorr.

A particularly suitable apparatus and method for producing laboratory devices, filtration or binding media in accordance with the invention is described in W02005/089961, the content of which is hereby incorporated by reference.

Precise conditions under which the plasma polymerization takes place in an effective manner will vary depending upon factors such as the nature of the polymer being deposited, as well as the nature of the substrate and will be determined using routine methods and/or other techniques.

The dimensions of the chamber will be selected so as to accommodate the particular substrate or device being treated. The chamber may be a sealable container, to allow for batch processes, or it may comprise inlets and outlets for the substrates, to allow it to be utilised in a continuous process as an in-line system. In particular in the latter case, the pressure conditions necessary for creating a plasma discharge within the chamber are maintained using high volume pumps, as is conventional for example in a device with a "whistling leak". However it will also be possible to process substrates at atmospheric pressure, or close to, negating the need for "whistling leaks".

The thickness of the deposited polymer coating which is applied using the method of the invention will depend upon the nature of the product. Generally, the thickness of the coating may be uniform, so as to ensure that binding of biomolecule takes place evenly all over the surface.

Factors which may be used to control thickness include the length of exposure to the plasma and the pattern of the pulsing, as well as the pressure, flow rate and nature of the monomer.

Generally, a coating of a sugar polymer which is up to 5000A thick, for example from 1-2000A is applied for most assay purposes, as well as the formation of biocompatible surfaces.

Using the method of the invention, a variety of devices equipment, including assay plates such as microarrays, models systems, sensors used in glycobiology, and medical devices such as implants, including oesteoimplants, anti-tumour implants, stents and the like may be prepared. They may be two or three-dimensional as required.

These devices obtained using the method form a further aspect of the invention.

Example 1 -Preparation of a Glycobiological Sensor A sensor substrate (for example a nitrocellulose membrane or silicon nitride on an AFM tip), is placed inside the appropriate sized vacuum chamber and evacuated to low pressure -5 mtorr. On reaching base pressure and ensuring the out-gassing rate is satisfactory, a galEMA and gas mix are introduced to a pressure of 80 mtorr. On reaching the required operating pressure a pulsed plasma is struck using a radio frequency source at 60W peak power at a pulse on-time of 50 ms and an off time of 250 ms and the deposition of a well adhered, thin plasma polysaccharide polymer ensues. The deposition process runs for 20 mins after which the RF, monomer and gases are turned off and the system is evacuated to base pressure. The resulting plate with a functionalised layer is then removed and is ready for use.

Claims (5)

1. Claims 1. A method for depositing a sugar molecule on a surface, said method comprising exposing a surface to a plasma containing a sugar monomer, under conditions in which the monomer is deposited on the surface so as to form a polysaccharide layer thereon.
2. 2. A method according to claim 1 wherein the sugar monomer comprises a monosaccharide, linked to a polymerisable group.
3. 3. A substrate having a polymeric sugar layer thereon, obtainable by a method according to any one of the preceding 15 claims.
4. 4. A substrate according to claim 3 which is a glycobiological sensor or model system.
5. 5. A substrate according to claim 3 wherein the sugar layer is a biocompatible layer, and the substrate is a biological implant.