JP2000351862A - Neutral coating film - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a composite material which has a wettable surface significantly made hardly susceptible to protein adsorption (staining) by forming, on the surface of a bulk material, a hydrophilic coating film which is covalently bonded to reactive groups on the surface and has pH-sensitive wettability- imparting groups partly modified with a neutralizing agent. SOLUTION: The composite material comprises a bulk material and a hydrophilic coating film. The film is covalently bonded, directly or indirectly through functional groups of an oligo-functional organic binding agent, to reactive groups on the surface of the bulk material and has many pH-sensitive wettability-imparting groups selected from among COOH, NH2, and SO3H groups, the pH-sensitive groups having been partly modified with a neutralizing agent selected from among organic acids and organic bases. Preferably, the reactive groups originally exist on the surface of the bulk material or are attached thereto by a plasma treatment. Preferably, the hydrophilic coating film is based on a polysaccharide or a (meth)acrylic acid (co)polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to a composite material comprising a bulk material and an antifouling hydrophilic coating, wherein the antifouling coating comprises a particularly neutralized polysaccharide. Also described are articles comprising the composite, particularly biomedical devices.

[0002]

BACKGROUND OF THE INVENTION Irreversible adsorption and subsequent denaturation (fouling) of proteins on biomedical devices has various deleterious consequences that must be overcome by improved biomaterial design. For example, in the case of contact lenses, fouling not only requires cleaning, but also limits the wearable time of the contact lenses, and more importantly, the adsorbed proteins cause adverse events. For example, proteins adsorbed on the surface, such as fibronectin,
It can act as a "base" for bacteria to adhere to. In the case of blood-contacting devices such as vascular grafts and synthetic heart valves, irreversible adsorption of proteins such as fibrinogen leads to platelet activation, clotting and thrombosis. Other biomedical devices, such as percutaneous and urinary catheters,
Can act as a place for bacterial infection. Again,
It is believed that the host proteins adsorbed on the device promote bacterial attachment.

[0003] Clearly, there is a need for biomaterials that have the ability to significantly reduce or completely eliminate irreversible protein adsorption. This need has been recognized in the biomaterials literature for many years and there are numerous publications and patents describing "non-fouling" or "anti-fouling" surfaces or coatings. However, in closer examination, researchers have measured the amount of adsorbed protein using a method that is not as sensitive as the highest level, or by comparing a new surface / coating with a given material. (In many cases, a highly adsorptive but widely used known material such as polyethylene or PVC is selected to adapt the effect!) The use of a comparative description is revealed. Usually, "non-fouling" or "anti-fouling" surfaces or coatings have been measured to adsorb proteins on the order of 300 ng / cm ² , approximately corresponding to a protein monolayer. Such ranges are usually achieved in 1-2 hours or less. Thus, there is still a need to create surfaces or coatings that can minimize protein adsorption over long periods of time.

[0004] EP 613,381 discloses a multilayer material comprising a polysaccharide coating. Such coatings have been found to have relatively low fouling properties as compared to known conventional hydrogels, such as poly-HEMA, and are useful in the production of contact lens coatings having good clinical performance (HJ Griesser et al., Polysaccharide Coatingsfo
r Contact Lenses, Polymeric Materials Science and
Engineering, 76, 79 (1997)).

[0005]

The present invention improves upon this and other prior art by disclosing a coating, especially a modified polysaccharide coating, which further reduces the amount of protein that can be adsorbed. Add. However, the concepts and chemical methods of reducing protein adsorption exemplified herein with respect to polysaccharide coatings can be transferred to other similar chemical coating structures to reduce protein fouling in various biological materials. Can be applied.

[0006]

SUMMARY OF THE INVENTION Accordingly, the present invention provides
It relates to the subject matter disclosed in claim 1. The neutralizing agent is an organic base in the case of the wetting group —COOH or —SO ₃ H, and an organic acid in the case of the wetting group —NH ₂ . As used herein, "at least partially" preferably means that at least 20% of such groups are modified, more preferably at least 50% of such groups, all calculated on a molar basis. Is modified, most preferably at least 80% of such groups are modified.

The present invention is also a method of improving the non-fouling properties of a hydrophilic coating on a bulk material, comprising -COOH or -COOH.
Treating the wetting groups in the coating selected from SO ₃ H at least partially with an organic amine or at least partially removing the wetting groups in the coating that are —NH ₂ groups from an organic acid; And a method characterized by performing the following.

[0008] In the case of contact lens fouling, lysozyme is the major fouling protein. In some cases, lysozyme is the only protein that makes up the first monolayer of adsorbed proteins that prepares the contact lens surface for further adsorption of various subsequent protein and lipid layers. Lysozyme associates with the negatively charged chemical groups of the contact lens material. Thus, contact lens coatings made from negatively charged polysaccharides, such as hyaluronic acid and chondroitin sulfate, are highly wettable and relatively non-fouling compared to many of the prior art, but have poor lysozyme adsorption. It does not have the ultimate resistance that is ultimately desirable.

Typical polysaccharides include hyaluronic acid, deacylated hyaluronic acid, chitosan, chitin 50, fucoidan,
Carrageenans, chondroitin sulfate, dextran, blue dextran, aminated dextran, carboxylalkylated dextran, glactomannan, pullulan,
From glycosaminoglycans, heparin, agarose, curdlan, pectin, pectic acid, xanthan, hydroxypropylcellulose or chitosan, carboxymethylcellulose or chitosan, emulsan, laminaran, inulin, pustulan, scleroglucan, schizophyllan and mucopolysaccharide Selected.

Preferred polysaccharides are hyaluronic acid, deacylated hyaluronic acid, chondroitin sulfate, dextran,
It is selected from blue dextran, aminated dextran and carboxylalkylated dextran.

"Alkyl" refers to groups having up to 18, preferably up to 12, and especially up to 7 carbon atoms.

The invention particularly relates to a polysaccharide coating in which the charged groups are blocked by a suitable chemical reaction to form an uncharged, so-called neutral coating. Such coatings,
Compared to the known polysaccharide coatings disclosed in EP 613,381, it has much better resistance to protein adsorption and, after 2 hours immersion in artificial tears, much less than a monolayer Accumulates only total protein material. The known polysaccharide coatings disclosed in EP 613,381 accumulate proteins of a monolayer mass (on the order of 300 ng / cm ² ) in the same time and possibly even shorter times (see below). See example).

"Uncharged groups and / or coatings" and "neutral groups and / or coatings" are used interchangeably in the present invention. Therefore, "charged" and "non-neutral" also
Use as a synonym.

The neutral coating of the present invention is produced by covalently anchoring polysaccharide molecules to a bulk material, preferably a bulk biomaterial. As described in EP 613,381, a polysaccharide molecule can be directly bonded to a bulk material by an interfacial reaction that creates one or more covalent bonds between the polysaccharide molecule and the surface of the bulk material. Alternatively, a thin interfacial bonding layer can be bonded in between. Direct bonding may be due to the bulk material having surface chemical groups suitable for covalent interfacial reactions, or if such reactive groups can be combined with known surface treatments described in the art, such as ammonia gas plasma ( It must be introduced by a radio frequency (rf) glow discharge) process. However, the application of an intermediate thin interfacial tie layer is often preferred.
The reason is that this method has often been found to give better coating quality (homogeneity and reproducibility) and adhesion of polysaccharide molecules. This is all European Patent 613,38
No. 1 discloses it in detail. The invention is characterized by a further subsequent step which increases the fouling resistance.

More specifically, for example, in EP 61
The highly wettable but less fouling resistant polysaccharide coatings produced according to US Pat. No. 3,381 are further modified by further chemical reactions that convert charged chemical groups to neutral groups. For example, the carboxylate groups of eg hyaluronic acid coatings (mostly deprotonated at pH values of biological media such as tears and blood) are usually neutralized by reaction with amines such as ethanolamine . This converts the carboxylate group to an amide group,
Provides additional hydroxyl groups that enhance the wettability of the coating. However, hydroxyl groups are not required to practice the present invention. The main steps involve the carboxylate group,
This is a step of converting into a group that is uncharged at a physiological pH value. Such groups are usually selected from amides and esters. Amide groups are particularly preferred. The amide group may be, for example, a known N-ethyl-N '-(2-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) / N
-Hydroxysuccinamide (NHS) reaction.

However, the invention is not limited to polysaccharides. Other carboxylate-containing coatings, for example, 90:10
It can be produced by covalent attachment to a biomaterial such as a polyacrylamide / polyacrylic acid copolymer. The copolymer is, for example, EDC that binds the carboxylic acid groups of the copolymer to surface amine groups under acidic conditions.
/ NHS reaction covalently attaches to the surface containing amine groups. However, even after raising the pH to a value applicable for physiological applications, the carboxylate groups persist on the surface of the coating. The reason is that it is not possible to react all carboxylic acid groups with amine groups for steric reasons and surface amine density. The remaining carboxylate groups can then be neutralized, for example, by an EDC / NHS reaction.

It should not be concluded that the present invention is only applicable to fouling events controlled by lysozyme.
Negatively charged proteins can also adsorb to negatively charged (eg, carboxylated) surfaces and coatings. This is due to ionic crosslinking, ie, Ca ²⁺
It is thought that this occurs when a divalent ion such as is used as an intermediate. Therefore, neutralization of carboxylate groups is expected to be beneficial even if the fouling is not dominated by lysozyme.

Also, the concept of surface / coating charge neutralization is not limited to carboxylate groups. Charged surfaces and coatings can include other groups, such as amines.
Amine groups are positively charged at physiological pH values. Thus, negatively charged proteins can ionically associate with such a charged amine surface, and positively charged proteins can also bind through ionic cross-links. However, if the amine groups are converted to neutral groups by, for example, known reactions, such as acetylation, the charge on such surfaces and coatings can be neutralized. One example is a coating formed by covalently attaching aminodextran to an aldehyde or carboxylic acid surface. Attachment consumes some of the amine groups, but the remaining amine groups of the aminodextran coating present a positive charge to the biological medium. Neutralization of positively charged groups is also an object of the present invention.

The significant reduction in the protein adsorption propensity of the coatings of the present invention reduces clinical problems due to the reduction of deleterious consequences expected to occur as a result of reduced fouling, eg, reduced bacterial colonization. However, it enables the manufacture of contact lenses suitable for long-term wearing.

The coatings of the present invention can be used for continuous wear contact lenses (EWC) of various polymeric materials (bulk), such as perfluoropolyether or silicone containing compositions.
L) Can be applied to materials.

It is an object of the present invention to provide coatings with a reduced rate of protein fouling and thus a reduced rate of deleterious biomedical consequences.

This problem has been solved by neutralizing the charged groups on the surface or the coating. "Neutral coating structure"
Within the terminology of the present invention, it refers to those having a microscopic meaning, that is, having no charged group. Macroneutrality resulting from charge cancellation in the zwitterionic structure is not involved in the neutralization.

Measurement of Protein Absorption The accumulation of proteins on surfaces and coatings can be measured by X-ray photoelectron spectroscopy, increasing the N signal indicating the amount of protein and decreasing the signal of the biomaterial when the biomaterial is covered by the protein. Can be measured by FIG. 1 is a graph showing an XPS analysis of lysozyme accumulation on a heptylamine plasma polymer coating. Initial data (time zero) represents the nitrogen content of the heptylamine plasma polymer coating, with an increase in nitrogen content signifying additional nitrogen by lysozyme. The sample was thoroughly rinsed after immersion in the protein solution. Thus, the amount of protein detected is indicative of tightly adsorbed, ie, irreversibly adsorbed protein. Although you might have expected that lysozyme would not adsorb to such amine-containing surfaces, fouling by lysozyme clearly occurs to levels in the range of about one monolayer (範 囲 220 ng / cm ² ) within 20 minutes. . The quantities calculated from these XPS data have been verified by the quartz crystal microbalance method. Therefore, methods other than "similar charge" must be performed to prevent adsorption of proteins of a given charge.

Similar rate plots of protein accumulation were obtained for the polysaccharide coating. Some of the coatings disclosed in EP 613,381 surprisingly showed a protein fouling rate, similar to the polymer coatings described above, but the polysaccharide coating did not Was very hydrophilic and had a high affinity for water, as indicated by its ability to retain DNA. Most notably, it was found that the most hydrophilic coating, the chondroitin sulfate coating tested according to the contact angle data, fouled at a faster rate than many other coatings.

FIG. 2 is a graph containing data from XPS analysis of polysaccharide coatings immediately after manufacture (control) and after immersion in artificial tears for one hour. Again, the amount of protein represents only the irreversibly bound protein. The finite nitrogen level of the control sample is due to the nitrogen-containing interfacial tie layer where the polysaccharide was immobilized. Therefore, as in FIG.
The increase in the N signal relative to the control is a measure of the amount of protein adsorbed.

Clearly, negatively charged coatings such as carboxymethylated dextrans (CM), chondroitin sulfate (CS), and hyaluronic acid (HA) adsorb significant amounts of protein in one hour. CS and HA adsorb in the monolayer range. Oxidized dextran (OD) is a coating that has no charge on the polysaccharide itself, but the covalent bond is an amine bond, and thus there is some positive charge. Obviously, a coating with a lower charge (C
M, OD) adsorb less protein in a given time. However, by far the best performing is the hyaluronic acid (EA / HA) coating blocked with ethanolamine. It adsorbs far less than one monolayer in one hour.

It is difficult to determine if the blocking reaction (converting the carboxylate groups to hydroxyethylamide) has taken place to an efficiency of 100%, and a small amount of the negatively charged carboxylate groups can cause the HA / EA coating May remain inside. However, obviously, the advantage of reducing the number of charged groups is significant.

Uses The present invention is not limited to contact lenses. Charge interactions are very important in many other biomaterial applications, and the production of neutral coatings leads to reduced fouling and, in many applications, reduced incidence of harmful biomedical consequences in many applications Is expected. A first object of the coatings according to the invention relates to blood-contacting applications (vascular grafts, heart valves, dialysis membranes) where the reduction of fibrinogen adsorption is very advantageous.

The coatings of the present invention are also useful as antimicrobial coatings for applications such as indwelling catheters.

Another area where uncontrolled protein adsorption reduces the efficacy of the device is in the field of biosensors. Specificity recognition entities, such as charge neutralizing coatings bearing antibodies, are expected to provide enhanced signal-to-noise ratios, thereby providing lower detection limits and more reliable assays.

[0031]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Plasma Polymer Surface A 25 mm thick, 12.7 mm wide fluorinated ethylene propylene copolymer (Teflon FEP) was purchased from Du Pont and used as a substrate for preparing functionalized surfaces. F
EP has many significant advantages over other materials that have been used as model substrates, such as polystyrene and glass. FEP does not readily adsorb hydrocarbon pollutants from the atmosphere and therefore does not require extensive cleaning. A clean surface also helps to accurately calculate the thickness of the overcoat layer, ensuring that a well-defined structure exists and that the elemental composition measured by XPS is close to the theoretical composition. All coatings used in this study did not contain fluorine, so the attenuation of the F1s signal of the FEP substrate was used to measure the thickness of the overcoat layer (see equation in the text). The smooth morphology of FEP as measured by scanning tunneling microscopy (STM) is based on the angular resolution XPS (ARXP
S) Facilitate its use in research.

2. Plasma Device A FEP substrate was coated with a plasma polymer film using a technique based on standard radio frequency (rf) glow discharge. Plasma polymerization was performed in a custom reactor. The reaction chamber consisted of a vertical glass cylinder 16.5 cm in diameter and 34 cm in height. A glow discharge was generated between copper electrodes 11 cm apart. The base electrode was circular with a diameter of 9.5 cm, on which the FEP piece for coating was arranged. The upper electrode was U-shaped (length 17 cm, width 8 cm) and was made of copper rod. The process vapor (monomer) gas inlet was located between two vertical electrode bars in the top plate used to seal the reaction chamber. The process vapor supply line consisted of a pressure control valve to control the flow and a bellow attached to a round bottom flask containing the monomer liquid. The outlet is a base plate (PTF
In E), it was located below the base electrode. Also, there was a separate gate valve at the bottom of the reaction chamber to vent the entire reaction chamber. The rf glow discharge is a radio frequency generator (ENI,
Power was supplied by model HPG-2). The system was evacuated using a Javac oil seal high vacuum pump. The pressure in the reaction chamber was accurately measured using a pressure transducer (MKS Baratron, model 127A).

4.2.3 Plasma Polymerization Conditions Before introducing the monomer gas, the pressure in the reaction chamber was reduced to a base pressure of 0.01 mbar or less. n-heptylamine (Aldric
h Chemical Co., using a ³ 99%) monomer, for subsequent polysaccharide immobilized, were coated with FEP substrates with a thin polymer coating containing amine groups. Typical plasma deposition conditions used were a power of 20 W, a deposition pressure of 0.12 mbar, and a frequency of 200 kHz. Deposition times of 3-5 seconds were used to obtain thin coatings (thickness <12 nm) suitable for thickness measurement by attenuation of the XPS F1s signal. The resulting coating was measured to be 3-4 nm thick. All other plasma depositions were performed for 20 seconds.

3. Polysaccharide coating Chondroitin sulfate (CS), NaCNBH3 (90
%) And N-ethyl-N '-(2-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) were all purchased from Sigma and used without further purification. Obtain NaIO ₄ a (99.8% +) and N- hydroxysuccinamide (NHS) from Aldrich and used as received. Carboxymethyl dextrans (CMD) were prepared by using the reaction of bromoacetic acid with dextran and used without further purification. Similarly, dextran (Sigma, M
W71,400 Da) was carboxymethylated by reaction with bromoacetic acid. Carboxymethyl dextrans (CMD) with different ratios of carboxy groups to anhydroglucopyranoside (Glc) ring units were prepared by using different ratios of dextran to bromoacetic acid to obtain different degrees of substitution. did.

3.1 Preparation of carboxymethyl dextrans 10 g of dextran was dissolved in 50 ml of 2M NaOH containing 0.125M, 0.25M or 1M bromoacetic acid. The solution was stirred overnight, dialyzed and lyophilized. The degree of carboxylation was determined by back titration and NMR spectroscopy. The experimentally determined ratio is 1: 2,
1:14 and 1:30. Different substitution ratios have made it possible to produce coatings with different surface deposition densities and thus different coating microstructures.

MilliQ H ₂ O (Millipore system, 1
Prepare all solutions using 8.2 M1 / 2 / cm)
Rinse all clean glassware.

4. CS, HA and CMD The newly applied n-heptylamine plasma polymer (n-happ) substrate is immersed in a 1 mg / ml aqueous solution (approximately 18 ml) of a suitable polysaccharide and shaken for 1 hour to obtain a polymer. Was made to reach a plateau. N-ethyl-
The addition of a 2.0 ml solution of N '-(2-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 19.2 mg / ml) and N-hydroxysuccinamide (NHS, 11.5 mg / ml) resulted in The carboxyl groups present were activated to form N-hydroxysuccinimide esters. Hyaluronic acid (HA) is n-hap
FIG. 3 shows a reaction formula for immobilization to p. The substrate was shaken overnight, rinsed three times with H ₂ O, and visually inspected for water wettability.

5. Hyaluronic Acid + Ethanolamine Neutralization A FEP / n-happ / HA-based test piece was immersed in a 6M aqueous solution of ethanolamine (NH ₂ (CH ₂ ) ₂ OH) for 1 hour, and then EDC was applied as shown in FIG. / NHS, shaken overnight, then rinsed three times with H ₂ O and visually inspected for water wettability. The reaction was designed to block residual carboxyl groups on the HA surface, potentially adsorbing oppositely charged proteins, as outlined in FIG.

7. Preparation of protein adsorption solution Lysozyme (chicken egg, 95%), lactoferrin (bovine colostrum, 90%), α-acid glycoprotein (bovine, 99%),
γ-globulin (human, 99%), human serum albumin (HSA, 97-99%), bovine mandibular mucin (BSM,
Type 1-S), trypsinogen (bovine pancreas), myoglobulin (horse heart, 95-100%), cholesteryl linoleate (98 +%), triolein (99%),
Oleic acid propyl ester (99%), dicaproin (99%, mixed isomer), undecylenic acid (Na salt, 9
(8%) and cholesterol (99 +%) were purchased from Sigma and used without further purification. Dodecyltrimethylammonium bromide (DTAB, 98%, BD
H Chemicals) and linalyl acetate (97%, Aldrich)
Was used without further purification. NaCl, K
Cl, NaHCO ₃ , lactic acid, CaCl ₂ and NaH ₂ PO ₄
H ₂ O was of analytical grade. 3- (N-morpholinopropane) sulfonic acid (MOPS) buffer is available from Calbiochem.
Purchased from the company. Analytical grade toluene, ethanol and n-hexane were purchased from BDH Chemicals.

8. Preparation of ATF Artificial tear (ATF) was prepared according to the prior art.
Table 1 lists all ATF components. The preparation procedure required the following series of steps.

Step 1. Buffer stock Prior to dissolving the proteins and lipids, a stock solution (about 250.0 ml) of buffer containing all salts was prepared. Finally, CaCl ₂ was added to the buffer solution to prevent precipitation of CaCO ₃ , which is insoluble at low ionic strength.

Step 2. Raw material lipid mixture Oleic acid propyl ester (0.30 g), triolein (0.40 g), dicaproin (0.08 g) and linalyl acetate (0.50 g) were combined and mixed by shaking. Next, by shaking and gently heating in a water bath, cholesterol (0.012 g), cholesteryl linoleate (0.18 g) and lipid mixture (0.4 g) were added.
08g) were mixed. The temperature was kept at a minimum (<50 ° C.) to prevent lipid volatilization. The raw lipid mixture was dried and stored at −18 ° C. under nitrogen.

Step 3. Mucin lipid mixture (approx. 200 ml) The final lipid mixture (0.016 g, step 2) and mucin (0.30 g) consist of solids and liquids in small beakers (approx.
0 ml) and homogenized by pressing with a flap. Three 1.0 ml portions of buffer were added to aid in the micellization of mucins and lipids. The generation of frictional heat was minimized to prevent mucin thermal denaturation and lipid volatilization. Undecylenic acid (0.003 g) is dissolved in 1.0 ml of buffer,
0.1 ml of this solution was added to the mucin lipid mixture. The final mucin lipid mixture was added to a volumetric flask to 200.0 ml. During the process, the residual mixture was thoroughly rinsed from the mixing beaker.

Step 4. Addition of protein The final ATF was prepared in 10.0 ml batches to ensure consumption before degradation or microbial growth. Each protein, lysozyme (95 mg), lactoferrin (95 mg), α-acid glycoprotein (25 mg), human serum albumin (10 mg) and γ-globulin were completely dissolved and 20 mg to prevent aggregation and foam formation. The mucin lipid mixture (approximately 50.0 ml) was added in the following manner.

[0045]

[Table 1]

The ATF was maintained at 4 ° C. overnight and 1N Na
After adjusting the pH to 7.40 with OH, the mixture was frozen at -18 ° C. ATF was thawed to room temperature (about 22 ° C.) before the adsorption experiments. The freeze-thaw step of a protein solution is very important to prevent its degradation.

9. Adsorption Method (Adsorption Method Containing ATF) A piece of the substrate was immersed in a 2.0 ml ATF solution for 1 hour, rinsed with H ₂ O three times, and then stored under H ₂ O until analysis to obtain all polysaccharides (PS). ) The adsorption behavior of the coating film to ATF was compared. In addition, OD and CS substrate pieces are
The solution was immersed for an adsorption period from 5 seconds to overnight (about 16 hours) to compare the time dependence of the adsorption. The OD substrate was also exposed to a modified ATF solution depleted of lysozyme for the same period. The CS substrate was immersed in the same solution for one hour.

10. Immobilization of HA, CS and CMD Wetting or hydrophilization is one of the essential standards required for the development of continuously worn contact lenses. As an alternative to OD, we attempted to meet this criteria using four other polysaccharides. CMD was prepared by converting a controllable portion of the hydroxyl groups of dextran to carboxymethyl groups with bromoacetic acid. Selecting two different CMDs, one CM / sugar ratio is 1/13,
Another CM / sugar ratio was 1/30. The principle was to vary the number of attachment points per monomer of dextran to form a coating with varying protein rejection ability based on the degree of "floppiness" at adjacent interfaces. The deposition of HA and CS takes advantage of the inherent structural chemistry and eliminates the need for further modification steps. Both structures are shown in FIG. Each of the chondroitin sulfate and hyaluronic acid monomer units consists of a glucuronic acid sugar unit containing one residual carboxylic acid functional group.

FIG. 3 outlines the attachment chemistry for immobilizing HA on the n-happp surface, and can also be applied to the CS and CMD surfaces. After allowing enough time (about 1 hour) for the polysaccharide to physically adsorb to the n-happp surface, the carboxyl group is reacted with EDC / NHS to form an active N-hydroxysuccinimide ester, It is replaced by a primary amine group on the n-ha surface to form a covalent amide bond with the polysaccharide molecule.

All polysaccharide grafts using the reaction scheme of FIG. 3 exhibited excellent visual water wettability when held in a vertical position. After preparation, the surface showed some water repellency, and the graft was sometimes discarded because it was considered patchy. Table 2 summarizes the XPS elemental composition of the three polysaccharides grafted using the EDC / NHS reaction mechanism and data for each surface after immersion in ATF for 1 hour.

First, when CS is attached, n-happ
For the surface case, the oxygen content increased from 4.0% to 18.5% and the carbon content decreased from 80.4% to 68.7%. Although this change is consistent with expectations based on the C and O contents of chondroitin sulfate, the composition of the coated sample is not the same as the composition of the pure CS, so for the OD coating, the thickness of the CS coating was It is immediately apparent that is substantially smaller than the XPS sampling depth. Although the underlying n-ha face does not contain sulfur, but because CS contains two S atoms per monomer unit in the form of a sulfonate, an S content of 0.8% of the grafted face may indicate a successful reaction. Provides further evidence (FIG. 5). Further, since the CS of N- acetylglucosamine group (-NHCOCH _3), where the 6.6% for N content control of the graft surface 7.
It increased to 9%.

[0052]

[Table 2]

For example, when CS is attached, n-hap
Compared to the p control, there is a 1.4 nm thickness increase calculated from the increased attenuation of the F1s signal due to the graft layer. Thickness is not a true representation of the actual thickness in an aqueous environment. The reason is that the CS polymer hydrates substantially when contacted with an aqueous medium, but dehydrates in the reduced pressure environment of XPS analysis.

11. Protein Adsorption from ATF to Polysaccharide Table 2 lists all XPS data for protein adsorption from ATF after one hour on the five polysaccharide surfaces, and FIG. 2 shows the results before and after immersion in ATF. 3 is a comparison plot of the N content of each surface including the HA block surface. All polysaccharide surfaces showed a significant increase in nitrogen signal corresponding to protein adsorption after immersion in ATF for 1 hour.

The CS and HA surfaces showed the highest levels of protein adsorption from the ATF solution on all polysaccharide surfaces tested. Nitrogen content on CS side is 7.9% of control
From, it increased to 15.1% after immersion in the ATF solution for 1 hour, and the N content of the HA surface increased from 6.9% to 13.9%. These increases in nitrogen content are consistent with approximately monolayer coverage by the adsorbed proteins. HA
The high number of carboxyl groups in large polymers of
a indicates that it cannot be covalently attached to the amine group on the pp face. Thus, there is likely to be a substantial number of residual carboxyl groups in the HA coating that give a negative charge at pH 7.4. These residual carboxylate groups were intended to be blocked with ethanolamine to study whether the negative charge of the HA coating was responsible for protein adsorption from ATF.

In order to reduce the amount of protein adsorbed from ATF, a step of inactivating (neutralizing) unreacted carboxyl groups remaining on the HA surface using ethanolamine was performed. XPS data ranged from 6.9% to 9.0% of N content.
To indicate that the reaction was successful (Table 2, FIG. 6). The O content is also 14.5%
And the thickness of the modified hyaluronic acid surface was calculated to be 3.1 nm compared to 2.7 nm for the original surface, further evidence that the blocking agent was bound to the HA surface. Offered.

The modified HA surface was immersed in ATF for 1 hour. X
The PS data suggests that there is a significant decrease in the amount of protein adsorbed on the surface, increasing the nitrogen content from 9.0% to 10.4%. This is 6.9 for the unmodified surface
% To an increase of 13.9%. The thickness of the unmodified surface increased from 2.7 nm to 5.8 nm, whereas the thickness of the blocked surface increased only from 3.1 nm to 3.6 nm. Due to the substantial reduction in the level of protein adsorption, a significant portion of the remaining negatively charged carboxyl groups in the HA matrix have been successfully blocked. That is, ATF
There is a reduction in the electrostatic attraction of proteins, such as lysozyme. However, the efficiency of blocking is unknown, and protein adsorption may result from any unblocked carboxyl groups or other interfacial interactions of ATF with the protein.

[Brief description of the drawings]

FIG. 1 is a graph showing an XPS analysis of lysozyme accumulation on a heptylamine plasma polymer coating. Initial data (time zero) represents the nitrogen content of the heptylamine plasma polymer coating, with an increase in nitrogen content signifying additional nitrogen by lysozyme.

FIG. 2 is a graph containing data from XPS analysis of polysaccharide coatings immediately after manufacture (control) and after immersion in artificial tears for 1 hour. CM (1/30) is a carboxymethylated dextran having a ratio of carboxymethyl groups to anhydroglucopyranoside (dextran) units of 1:30, and CM (1/13) is a carboxymethyl group and dextran units. Is a carboxymethylated dextran with a ratio of 1:13, CS = chondroitin sulfate, HA = hyaluronic acid, and HA / EA means hyaluronic acid that is at least partially modified by ethanolamine. And OD = oxidized dextran.

FIG. 3 shows the binding of hyaluronic acid (HA) to amino groups contained on the surface of a fluorinated ethylene propylene copolymer (Teflon FEP) plasma-coated with n-heptylamine (n-happ), which is activated Ester (N
-Hydroxysuccinimide ester).

FIG. 4 is a formula showing the modification (neutralization) of the remaining carboxyl groups of hyaluronic acid (HA) by an organic base (here, ethanolamine).

FIG. 5 is a formula showing the structure of hyaluronic acid (HA) (top) and the structure of chondroitin sulfate (CS) (bottom).

FIG. 6: AT on heptylamine plasma polymer coating
4 is a graph showing XPS analysis on F protein accumulation.

Continuation of the front page (71) Applicant 591269435 Commonwealth Scientific and Industrial Research Organization COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZAT ION Australia 2601 Australia Capital Territory No. Country Victoria 3792 The Patch View Road 20 (72) Inventor Peter Kingshot Germany 52062 Aachen Belkdulish 2a

Claims (14)

[Claims] 1. The method of claim 1, further comprising the step of reacting the surface of the bulk material with a hydrophilic coating, either directly or indirectly via the functional groups of an oligofunctional organic binder, according to the prior art. Is attached covalently to a functional group, and the hydrophilic coating film is-
COOH, including a number of pH sensitive wettability imparting group selected from the group consisting of -NH ₂ and -SO ₃ H, in a composite material having hard wettable surfaces suffer protein adsorbing (fouling) significantly, the wet resistance A composite material wherein the imparting group is at least partially modified by a neutralizing agent selected from organic acids and / or organic bases. 2. The method of claim 1, wherein the reactive groups on the surface of the bulk material are originally present or attached to the surface of the bulk material by plasma surface treatment.
The composite material as described. 3. The composite material of claim 1, wherein said hydrophilic coating is selected from the group consisting of polysaccharides, (meth) acrylic acid homopolymers and (meth) acrylic acid copolymers. 4. The composite of claim 1, wherein the oligofunctional organic binder is a bis-oxirane, diisocyanate, dicarboxylic acid chloride, ditosylate, dimesylate or epihalohydrin. 5. The composite material according to claim 1, which is a biomedical device, an ophthalmic device or a contact lens, preferably an ophthalmic device or a contact lens, more preferably a contact lens. 6. The organic acid is a carboxylic acid such as formic acid, a linear or branched alkane carboxylic acid such as acetic acid or propionic acid; an aryl carboxylic acid such as benzoic acid; The composite material according to claim 1, wherein the alkanesulfonic acid is selected from the group consisting of methanesulfonic acid and arylsulfonic acid, such as benzenesulfonic acid and toluenesulfonic acid. 7. The organic base is a secondary amine, for example, a linear or branched dialkylamine which is unsubstituted or substituted by one or more hydroxy groups in the alkyl chain; and Primary amines are selected from, for example, straight or branched chain alkylamines, which are unsubstituted or substituted by one or more hydroxy groups in the alkyl chain, wherein the alkyl is a carbon atom. Composite material according to claim 1, which refers to a group having up to 18, preferably up to 12, and in particular up to 7, carbon atoms. 8. The composite of claim 1, wherein the hydrophilic coating is a polysaccharide and is selected from dextran, carboxymethyl dextran, chitosan, hyaluronic acid, mucin, fucoidan, chondroitin sulfate, and glucosamine. 9. The composite of claim 1, wherein the modification of the wetting group comprises a multi-step reaction. 10. Use of a neutralized wetting group in reducing surface protein adsorption of the hydrophilic polymer coating of claim 1. 11. Use of an organic base and / or an organic acid in neutralizing a wettability-imparting group contained in the hydrophilic coating film according to claim 1. 12. Use according to claim 11, wherein the neutralization is charge neutralization and the wetting group is a protonated or deprotonated ionized group. 13. A method for producing a composite material having a wettable surface according to claim 1 which is significantly less susceptible to protein adsorption (fouling), wherein the bulk material, preferably in its desired final form, is reacted with Exposing the reactive group to a low-pressure plasma in a vapor of at least one organic and / or inorganic compound under conditions in which a thin film containing groups is deposited on the surface of the bulk material; , At least two groups capable of chemically reacting with the activating group and / or the reactive group
Reacting with an oligofunctional compound having three functional groups or with an activated reactive group having at least one further functional group capable of chemically reacting with a hydrophilic coating material, eg, a polysaccharide; Reacting the reactive group or functional group with a hydrophilic coating material, for example, a polysaccharide; and reacting the surface-immobilized hydrophilic coating material with a neutralizing agent selected from organic acids and / or organic bases, especially Treating with a neutralizing agent. 14. A method for producing a composite material according to claim 1 having a wettable surface which is significantly less susceptible to protein adsorption (fouling) and wherein the reactive groups are originally present in the bulk material. Reacts the reactive group originally present in the bulk material with an activating group and / or an oligofunctional compound having at least two functional groups capable of chemically reacting with the reactive group, or a hydrophilic group. Reacting with an activating reactive group having at least one additional functional group capable of chemically reacting with the polysaccharide, e.g., a reactive coating or functional group on the surface; For example, a step of reacting with a polysaccharide, and a step of treating the surface-immobilized hydrophilic coating material with a neutralizing agent selected from organic acids and / or organic bases, particularly a charge neutralizing agent. To be Method.