JP2002537025A - Biocompatible materials with new functions - Google Patents

Abstract

The present invention relates to the field of biomaterials, i.e., materials used for contact with living tissue and biological fluids for prosthesis, treatment, storage, and the like. More specifically, the present invention relates to a novel approach to creating a biocompatible surface, wherein the surface can functionally interact with a biomaterial. The biocompatible surface comprises at least two components, such as a hydrophobic culture substrate and a macromolecule of hydrophilic character, which together form a novel biocompatible surface. A novel approach is based on contacting this hydrophobic culture substrate with a laterally patterned monolayer of hydrophilic, flexible macromolecules that exhibit a significant exclusion volume. The surface is polar and morphologically a molecularly heterogeneous surface. The structural characteristics of the macromolecular monolayer (eg, the thickness of the layer or its side density) include (i) the structural characteristics of the layer forming the macromolecule (eg, its molecular weight or its molecular structure), and (ii) the monolayer. It is determined by the method of creating the molecular layer (eg, by physical or chemical adsorption or by chemical bonding of macromolecules). The structural characteristics of layer-forming macromolecules are determined by synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of biocompatible materials, ie, materials used in contact with living tissue and biological fluids for prostheses, therapeutics, storage or other uses. is there. The working environment of any biomaterial is either a biological fluid or living tissue, and events that occur at the contact interface play a critical role in the overall performance of the biomaterial.

[0002] Many conventional biomaterials lack the ability to properly interact with or support the biological material that comes into contact with the biomaterial, causing undesirable biological reactions. cause. However, these undesirable reactions can be controlled by altering the chemical and physical properties of the surface of the biomaterial. In this regard, surface modification is a representative example of a well-known strategy for providing suitable biocompatible materials. The present invention teaches a novel approach to creating biocompatible material surfaces that can functionally interact with biological materials.

BACKGROUND OF THE INVENTION [0003] We consider that when biological and synthetic materials interact with each other, it is usually associated that they are not part of a biological environment, such as the human or animal body. Must. Biocompatible materials have been defined as materials that do not elicit an acute or chronic inflammatory response when interacting with biological materials and do not prevent proper differentiation of the peri-implant tissue ⁽¹⁾ .

Further, according to another current view, biocompatible materials i) control or induce cell growth and tissue organization, and ii) promote cell-cell or cell-tissue interactions. ⁽²⁾ iii) isolating the transplanted cells from the host immune system ⁽³⁾
And iv) the production and / or secretion of cell products can be regulated. However, according to this definition, many synthetic materials used as biomaterials are not biocompatible, and much effort has been made to find ways to improve the biocompatibility of these materials. ing.

[0005] One important example for the interaction of biological and synthetic materials is the adhesion of human or animal cells to a polymer substrate. Cell adhesion, for example, fibronectin (FN), which is adsorbed on the surface of a synthetic material, resulting in contact between said surface and adherent cells
And it is known to contain various adhesive proteins such as vitronectin (VN) ^{(4, 5, 6, 7, 8)} . These interactions are further provided by receptors through specific membranes belonging to the integrin family of cell adhesion molecules ^(9,10) .

[0006] The adsorption of proteins from biological fluids onto polymer surfaces depends on the physico-chemical properties of the polymer surface ^(5,15) . For example, it is well known that adhesive proteins adsorb in large amounts on hydrophobic polymer surfaces, but their adsorption, mainly due to hydrophobic interactions, results in conformational changes and consequently (See FIG. 1) ^(7,12) . These conformational changes in the adhesion protein are indicative of a reduced or eliminated interaction between the adhesion protein and the cell ^(8,13) , resulting in a reduced polymer-cell interaction.

[0007] Furthermore, the conformational change of proteins adsorbing on the surface of a synthetic material, for example on the surface of an implant or a medical device, may increase the thrombogenesis of said material, or foreign and continuous rejection of the implant or medical device Can also cause

[0008] Synthetic polymers are a class of materials that are often used as biocompatible materials with selection criteria based on their mechanical properties, stability, and ability to produce predetermined or required shapes and / or morphologies. It is. However, these materials are often not biocompatible. For example, the synthetic polymers currently used for the production of membranes with controlled permeability, such as polysulfone, polyester or polypropylene, are often not suitable for immobilizing tissue cells. This is because the function of these cells cannot be maintained for a sufficiently long time for the reasons mentioned above.

[0009] However, the biocompatibility of any substrate depends on the chemical and physical properties of said substrate.
It is possible to control by changing. Surface modification is suitable biocompatible
It is a representative example of a well-known strategy for providing materials. Thus, the polymer surface may be, for example, charged side groups added to the polymer backbone, ⁽¹⁴⁾ Adsorption or covalent attachment of biologically active proteins and peptides to polymers
Shape fixation⁽¹⁶⁾The surface texture or morphology of the polymer and the polymer matrix^{(17, 18)}Through
Denatured.

[0010] Surface modification is known to be of particular interest when initiated under mild conditions, such as, for example, photo-transplantation ⁽¹⁹⁾ , for example when performed using a selective reaction. Using selective reactions, it is possible to determine and / or preserve the shape of the substrate, including the macroporous or microporous structure as well as the mechanical properties. Examples of surface functionalization include that it can repel cells by i) charge ⁽³⁰⁾ , or hydrophilic / flexible ⁽²¹⁾ , or that ii) cells can be, for example, on hydrophobic surfaces. Macroscopically homogeneous macromolecules that can be attached through the conditioning of the protein or through the attachment of the protein to a peptide that mimics the binding region of the adhesive protein ^{(14, 21)} or an available linkage Surface.

[0011] μm- pattern features of a scale is not only patterns with no cell areas, are effectively adapted to create a pattern that can be attached to cells ^{(2 2).} In addition, sub-μm scale patterns created by, for example, mixtures of adhesive peptides and charged groups ⁽²³⁾ are also suitable as supports for cell culture. Light-implantation ^{(24, 25) in} combination with photographic printing techniques is an established way to achieve such patterns.

An important universal rank of surface modification is adhesion of the polymer to the underlying surface. Many
In many cases, these macromolecules exhibit hydrophilic properties, so that in biological environments solvent
However, the surface of the lower layer has hydrophobic characteristics, for example. The adhesion of the polymer is i
) Physical adsorption of amphiphilic polymer, ii) Use of self-assembled monolayer (SAM)^(26,27), Iii
) Surface binding of charged polymers ionic binding to opposite charge, iv) either light- or heat-
Transplantation of reactive polymer^{(24, 28)}, And when the underlying surface is a polymer,
, V) enranglement of said polymer into the polymer surface ( ^{(29, 30)} Also see).

In order to remove proteins coming into contact with the surface, and thus also biological materials such as cells, the surface of the hydrophobic base polymer is, for example, a layer or chain of (attached) hydrophilic macromolecules. It is known to coat and protect with. In particular, the protective properties of poly (ethylene glycol) (PEG), an established hydrophilic polymer, are well known. It has been agreed that the protein defense properties of PEG-coated substrates are driven by steric stabilization, which is induced by the excluded volume of the attached polymer ⁽³¹ , ³²⁾ . It is recognized as a combination of several molecular structures ⁽⁴⁾ .

[0014] The protein protection properties of the PEG-coated substratum show a correlation between the amount of protein adsorbed and the lateral density (the higher the lateral density of the attached PEGs, the lower the protein adsorption). It has been found that it depends on their lateral density on the surface ^{(33, 34 )} . According to the above, different techniques are known which describe how to make a coating base layer in which the coating effectively protects the underlying substrate, said coating also being able to be laterally patterned.

Recently, Sofia et al. ⁽³³⁾ , PEG grafted (molecular weight (MW): 3, 4, 10, and 20 kilodaltons (kDa)) protein (FN, cytochrome-c, and albumin) adsorption on silicon surfaces Was characterized over the area of the graft layer. Furthermore, the amount of grafting of the PEG moiety could be measured by photoelectron spectroscopy (ESCA). Protein adsorption decreased with increasing PEG graft density for all proteins and decreased by more than 95% for the highest PEG graft density compared to the silicon substrate without denaturation. From the measurement of the thickness of the adsorbed protein layer, the rod-shaped FN has its major axis along the surface for all PEG graft densities, and lie down on the surface by close contact with the surface. It was estimated to be adsorbed.

Thus, the protein can penetrate through the PEG layer and be effectively adsorbed to the surface of the lower layer from within or between the graft chains. Sofia et al. Found that protein adsorption began to decrease with respect to graft density when the grafted PEG chains began to partially overlap, and protein adsorption caused their free It was calculated to be negligible when limited to about half the volume. This correlation between protein adsorption and partial overlap of PEG chains is found for all investigated PEGMWs, and calculations have been performed to determine whether the grafted PEG chains have the same radius of gyration or spatial dimension as their solvated state. Based on the assumption that

Contact angle (CA) is commonly used to determine the wettability characteristics of a surface. The wettability of a surface is related to its hydrophilicity, as it is constituted by the components forming said surface. It is a very fine technique with a probe depth of about 5-10 Angstroms. CA is determined by measuring the angle defined by the phase limit of the liquid phase at the three phase boundary (solid / liquid / vapor). The three-phase boundary may be, for example, a vapor bubble in a liquid when the bubble is moored by the test surface (an anchored foam method, see also FIG. 2), or a droplet in the vapor placed on top of the test surface ( Droplet method). When polar (hydrophilic) test liquids (such as water) are used, hydrophilic test surfaces produce small angle values, such as 0 ° to 90 °, while hydrophobic test surfaces produce 90 ° to 180 °.
This produces large angle values of °. Both forward and backward measurements of CAs are used to characterize biocompatible materials according to the present invention ⁽³⁵⁾ .

Although the present invention is not intended to be limited by an understanding of the nature of any particular mechanism, or any particular related physical force, a single drop of liquid remaining on the substrate may include three forces: a) a solid And b) interfacial tension between liquid and vapor, and c) interfacial tension between liquid and vapor. The angle in the liquid phase is known as the "contact angle". BC Nayar and AW Adamson, “Contact Ang
le in Industry "Science Report, pp. 76-79 (February 1981). It is the angle contained between the tangent lines to the liquid surface and the tangent plane to the solid surface at points along their line of contact.

It is often found that advancing and receding contact angles are not the same. This hysteresis can be due to the chemical heterogeneity of the rough surface or substrate. One method of contact angle measurement is by taking a picture of the foam moored by the substrate and then measuring the angle from the picture. The angle can also be measured from a magnified image of the bubble. Low power microscopes produce sharply decoded images of liquid bubbles that are only observed as silhouettes through the eyepiece.

There are commercially available goniometers with environmental chambers with temperature and pressure control conditions in which the contact angle can be determined. It is also possible to attach the camera to such a goniometer. Commercial equipment for measuring contact angles is RAME-HART,
Inc. (Mountain Lakes, NJ), KRUSS (Charlotte, NC), CAHN INSTRUMENTS
(Cerritos, Calif.), And companies such as KERNCO INSTRUMENTS (El Paso, Tex.).

The contact angle ranges from zero (0) to 180 degrees (although the latter has not been experienced in practice). In the present invention, water is used as a probe liquid (representing the liquid phase), and air saturated with water vapor is used as a gas phase (forming bubbles). When the contact angle is between about 0 and about 90 degrees, the substrate is considered hydrophilic. 90 degrees is "intermediate aqueous". When the contact angle is greater than 90 degrees, the substrate is considered hydrophobic.

The elliptically polarized light method both changes the polarization state of light upon reflection from the surface of the base layer.
Based on the measurement, i) the refractive index of the bare surface, or ii) the film / coating on the substrate
Optical in-situ technology for measuring thickness and refractive index. Thus, high
The measured thickness and refractive index of the adsorption layer of the molecule can be converted to the value of the adsorption mass.
it can⁽³⁶⁾. In this way, for example, the interface of a polymer from a solution with that solution
It is possible to monitor the adsorption on the surface of the underlying substrate online. Of this method
Physical principle⁽³⁷⁾And assembly of equipment⁽³⁸⁾There is a detailed description of.Study of cell adhesion  The nature of the interaction of the surface of the synthetic material with the biological material, i.e. the material surface
Biocompatibility is related to the behavior of living cells when in contact with the surface
You. Therefore, criteria such as the amount of adherent cells, total cell morphology, cell surface mobility,
Localized adhesion formation, intercellular matrix (ECM) formation, and cell growth on material surfaces
Is important when monitoring and controlling the biocompatibility of material surfaces in vitro.
It is considered important.

The description identified below is believed to constitute the closest prior art relevant to the present invention. The prior art teaches how to protect polymers from biological materials, but does not address the technical problems solved by the present invention. Sofia et al. (1998), Macromolecules 31, 5059-5070, compare different PEG-type molecules and their interaction with proteins when chemically grafted onto a polymer substrate. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

US Pat. No. 5,776,748 relates to a device comprising a number of cytophilic islands and cell non-affinity regions made by self-assembled monolayers exhibiting cell-affinity or cell-affinity end groups. Cell adhesion may be promoted on cell-affinity or cell-affinity regions, respectively, by known mechanisms such as, for example, polar groups, charges and other introductions,
Or inhibited, but does not disclose binding of biological material in an active form.

US Pat. No. 5,002,582 relates to a method of producing a biocompatible material having an “effective” solid surface characterized by the properties of a hydrophilic polymer rather than a solid hydrophobic surface (column 8). The claimed biocompatible material does not have a contact angle that is substantially similar to that of a solid surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form. U.S. Pat. No. 4,973,493 relates to a method of creating a solid surface that is effectively protected by a biocompatible agent. The claimed biocompatible material probably does not have a contact angle that is substantially similar to that of a solid surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

US Pat. No. 4,722,906 relates to a method for selectively attaching a specific molecular target moiety covalently to a chemical moiety or substratum. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form. U.S. Patent No. 5,128,170 relates to a method for manufacturing a medical device having a surface that is highly biocompatible. The claimed biocompatible surface does not have a contact angle that is substantially similar to that of a medical device. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

US Pat. No. 5,728,437 relates to an article comprising a hydrophobic surface coated with a blood compatible surface layer. The coated surface does not have a contact angle that is substantially similar to that of a hydrophobic surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form. U.S. Patent No. 5,380,904 relates to a method for rendering a surface biocompatible.
A biocompatible surface does not have a contact angle that is substantially similar to that of an untreated surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

US Pat. No. 5,512,329 relates to a method for attaching a polymer to the surface of a substrate by applying an external stimulus. The method of claim 14 is directed to a method of modifying the surface properties of a substrate. No biocompatible material comprising a polymer determinant and a first determinant capable of bringing a polymer and a second determinant into contact with the first determinant is disclosed. The description also does not disclose attachment of the biological material to the disclosed polymeric material in an active form.

US Pat. No. 5,217,492 relates to a specific method for attaching biocompatible molecules to hydrophobic surfaces. The disclosed method for deposition is not relevant to the present invention. The description does not disclose attachment of the biological material to the disclosed polymeric material in an active form. U.S. Patent No. 5,263,992 relates to a biocompatible device that includes a solid surface and biocompatible agents that are located sufficiently close together to effectively protect the solid surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

[0030] US Pat. No. 5,741,881 relates to a bioactive coating capable of linking a particular polymer to a bioactive agent utilizing a hydrophilic spacer having a functional end group. The present invention does not utilize a bifunctional linker in the form of a hydrophilic spacer as a method for attaching the first determinant to the polymer substrate. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

WO 97/46590 relates to a support wherein the second outer layer is a hydrophilic polymer and a material comprising two layers, further comprising an immobilized biological material. The surface obtained by coating the support with a polymeric surfactant and a hydrophilic polymer does not have a contact angle that is substantially similar to that of the support. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form. WO 97/18904 relates to a method for providing a hydrophobic surface with a hydrophilic coating. The surface created by the hydrophilic coating does not have a contact angle that is substantially similar to that of a hydrophobic surface. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

[0032] EP 633031 A1 relates to compositions which can effectively protect polymers from biological materials. The protected polymer does not have a contact angle that is substantially similar to that of the unprotected polymer. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

Park and Griffith (1998), J. Biomat. Sci. Polym. Ed. 9, p. 89-110 provide a scaffold for specific PEG-PPO-PEG copolymers that can effectively inhibit cell adhesion. Has been disclosed. Copolymers are useful in controlling the three-dimensional organization of various cell types. Adhesion is achieved by the covalent attachment of a cell-specific carbohydrate ligand to the polymer that is capable of binding a particular receptor moiety. The present invention
It does not relate to the polymer substrate in contact with the first determinant. The cell adhesion properties of the biocompatible material according to the invention are determined, at least in part, by the cooperativity of the polymer substrate and the polymer, and optionally by a first determinant. The polymer "backbone" of the present invention is not itself cell-incompatible, as in the cited references. The disclosed copolymer materials are not relevant to the present invention and the description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

Nob et al. (1998), J. Biomat. Sci. Polym. Ed. 9, pp. 407-426 disclose modifications of PTFE membranes that substantially alter the contact angle. The disclosed biocompatible material is not relevant to the present invention, and the description does not disclose binding of the biological material to the disclosed polymeric material in an active form. Malmsten et al. (1998), J. Coll. And Interface Science 202, pp. 507-517,
We are testing the effect of chain density on protein adsorption inhibition. The description does not state the properties of the binding protein, and the description does not disclose binding of the biological material to the disclosed polymeric material in active form.

[0035] Zhang et al. (1998), Biomaterials 19, pp. 953-960, disclose a silicon surface modified with a PEG membrane to reduce protein adsorption. The silicon surface does not have a contact angle that is substantially similar to that of the PEG coating material. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form. Herbert et al. (1997), Chemistry and Biology 4, p. 731-737 discloses a method for identifying cross-linking of a bioactive molecule to the surface. No biocompatible material according to the present invention is disclosed, and the disclosed method is not relevant to the present invention since it is recognized that light-reactivation forms part of the prior art. The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

[0036] Wesslen et al. (1994), Biomaterials 15, p. 278-284 disclose the surface modification of hydrophobic polymers by the use of hydrophilic polymers including PEG. Denaturation significantly changes the contact angle (Table 1) and results in a decrease in polypeptide adhesion. The disclosed biomaterial is not relevant to the present invention, and the description does not disclose the attachment of the biological material in the active form to the disclosed polymeric material.

Bergstrom et al. (1992), J. Biomedical Materials Research 26, pp. 779-79.
No. 0 discloses polystyrene containing PEG bound in a compact, covalent form that can effectively reduce fibrinogen adsorption. Polystyrene does not have a contact angle that is substantially similar to that of a compact PEG surface.
The description does not disclose the attachment of the biological material to the disclosed polymeric material in an active form.

[0038] Desai and Hubbell (1991), Biomaterials 12, p. 144-153, disclose the incorporation of PEG and similar water-soluble polymers on the surface of biomedical polymers such as, for example, PET. The incorporation significantly changes the contact angle as shown in Table 1. The disclosed biomaterial is not relevant to the present invention, and the description does not disclose the attachment of the biological material to the disclosed polymeric material in active form.

Gombotz et al. (1991), J. Biomedical Materials Research, 25, pp. 1547-15.
62 discloses denaturation of PET surface with PEG. Incorporation is illustrated and considered the first
Change the contact angle significantly, as described in the chapter. The disclosed biomaterial is not relevant to the present invention, and the description does not disclose the attachment of the biological material to the disclosed polymeric material in active form.

SUMMARY OF THE INVENTION The present invention teaches a novel approach to creating a biocompatible surface that is capable of functionally interacting with a biological material. The biocompatible surface comprises at least two components, such as a hydrophobic substrate and a polymer of hydrophilic nature, which together form a novel biocompatible surface.

A novel approach is based on contacting the hydrophobic substrate with a laterally patterned monolayer of the hydrophilic and flexible polymer exhibiting a significantly excluded volume. I have. The two-component surface thus formed is a surface that is molecularly heterogeneous with respect to polarity and morphology. The structural characteristics of the polymer monolayer (such as, for example, the layer thickness or its lateral density) include: i) the structural characteristics of the layers forming the polymer (eg, their MW, or their molecules). And ii) the method of creating the monolayer (eg, by physically or chemically adsorbing or chemically bonding the polymer). And again, polymer (
The structural characteristics of the layers forming the (including plurality) are determined by synthesis.

The amount and conformation of biological material (such as, for example, polypeptides) that come into contact with the novel biocompatible surface, and thus also the biological activity, is also enhanced by the underlying hydrophobic substrate and polymer layers Determined and maintained by action. In this regard, it is possible to maintain and control the biological interaction between the contact polypeptide and other biological compounds such as cells and antibodies. Accordingly, the present invention aims to reduce and / or eliminate deactivation and / or denaturation associated with contacting a polypeptide and / or other biological material with a hydrophobic substrate surface.

In a preferred hypothesis, the solvated polypeptide penetrates the laterally patterned monolayer of the macromolecule, effectively adsorbs between the macromolecules of the macromolecule, and binds to the underlying hydrophobic surface. Adsorb. The polypeptide induces a lateral pressure acting between the macromolecule and the osmotic polypeptide as well as between the macromolecule itself to penetrate the monolayer of the macromolecule, causing the self-assembled macromolecule to penetrate. Must be deformed to some extent (
See also FIG. 3). This lateral pressure results from an undesirable loss of conformational entropy of the binding polymer, which is related to the spatial deformation of the polymer.
Thus, as the amount of osmotic polypeptide increases, the lateral pressure also increases.

According to the hypothesis, until a good balance in energy is obtained between i) unwanted induced lateral pressure and ii) favorable adsorption of the polypeptide to the underlying hydrophobic surface. , The amount of adsorbed polypeptide continues to increase. Thus, the polypeptide will continue to penetrate the polymer layer while effectively adsorbing to the underlying hydrophobic surface until the lateral pressure induced by that layer effectively repels any other polypeptide from that layer I do.

According to this hypothesis, the polypeptide adsorbed in and between the self-assembled polymer layers is exposed to lateral pressure resulting from the surrounding deformed polymer. The lateral pressure acting on the adsorbed polypeptide effectively protects the polypeptide from cleavage / denaturation and stabilizes the polypeptide in its active conformation, allowing it to be adsorbed but biologically active To produce a novel polypeptide.

Thus, the attachment, expansion, growth and tissue formation of cells on a surface depends on the biologically active polypeptides present on the surface, and thus, for example, to allow their control, The present invention solves the problem of how to provide surface design principles and modification methods-in a simple and inexpensive way. Thus, these new biocompatible surfaces can be used in cell culture dishes, bioreactors, implants, and biohybrid organs such as pacemakers without the need for further new polymer development and biocompatibility testing It is.

Thus, the present invention contemplates providing a means of creating a biocompatible surface suitable for use in emerging technologies, such as, for example, assembling and applying new surface structures of biocompatible materials with innovative functions. Can be Thus, the present invention is useful for the production of surface structures for use in biohybrid organs such as, for example, an artificial living pancreas, liver or kidney. The present invention allows the use of improved membranes, for example, to ensure that exogenous and / or allogeneic cells are spatially separated from the host immune system.

Modifying the membrane with the polymer layer containing a hydrophilic polymer such as PEG, for example, reduces the amount of protein adsorbed on the membrane surface ^{(24), while} simultaneously reducing FN and other It can improve the conformational / functional state / morphology of adsorbed proteins, such as attachment proteins.

The present invention also contemplates providing an array for culturing “sensory” cells, such as, for example, nerve, olfactory, retina and similar cells. Culture of sensory cells requires spatially dissipated reception of signals that must be organized in a highly complex and specialized manner. The signals emitted by those cells must dissipate in time and be transmitted to the non-biological support in a position-dependent manner. Photographic printing techniques, including for example the immobilization of PEG spacers and biospecific ligands, can be used to contribute in a highly specialized way to the structuring and / or functionalization of solid supports. It is anticipated that such a structure could eventually be used, for example, as a sensor or a biohybrid organ.

The cells which can be immobilized on the biomaterial according to the invention preferably have their function i) controlled delivery of biologically active substances such as, for example, hormones, ii)
Predetermined proteins and polypeptides derivable therefrom, such as antibodies, growth factors, and matrix factors, or iii) cells containing metabolites, preferably toxic or cytostatic metabolites, It is not limited to.
Examples of these types of cells are, for example, islets of Langerhans, hybridoma cells, chondrocytes, and hepatocytes.

The present invention is believed to be useful in organizing cells in organs and tissues. Such organization involves the controlled coordination of different types of cells connected on a micrometer scale through a local, highly organized network of different types of cells. It is envisioned that the present invention will allow photographic printing technology to be applied to the immobilization of macromolecules with foreign functional groups and biogenic ligands. The biomaterials thus created can immobilize them in a controlled and / or spatially assembled manner to allow for controlled coordination of different types of cells.

Having a particular functional group, such as a stochastically distributed macromolecule on a solid support (eg, itself or an amine group of either a biological or biological pseudoreceptor, or Without) to acquire the body of cells within organs and tissue-like structures, and subsequently, to form secondary clusters in the formation of clusters of different sizes (eg, clusters having different lengths with respect to the axis, eg, z-axis). It is also conceivable to use ligands (eg different macromolecules with different functional groups, for example functional groups affected by different chain lengths), and / or functional groups. In this way, the invention makes it possible to obtain a three-dimensional patterning of a given substrate. This is ultimately achieved by binding ligands that are selective for specific cell surface receptors, such as integrins and growth factor receptors, thus providing a structure for immobilization of, for example, single type cells, or, for example, co-cultured heterologous cells. To provide the possibility of providing a textured surface.

The new and innovative applications described hereinabove cannot be realized by the currently available state-of-the-art means, because of the lack of useful design principles and suitable methods for surface modification. Also, state-of-the-art means are not readily applicable to fine-tune the surface structure and / or biocompatibility of known polymeric biocompatible materials. The invention described herein represents a significant improvement over the state of the art and allows for the creation of potentially novel biocompatible materials and cell-based technologies.

According to one preferred embodiment of the invention, the surface of the biocompatible material has a contact angle that is substantially the same as the contact angle of the underlying hydrophobic underlayer of said surface. A substantially identical contact angle in the sense of the present invention is less than 4.5 degrees, e.g. less than 4.0 degrees, 3.7 degrees.
Including any contact angle variation within a number of values less than 5 degrees, such as less than 3.3 degrees, less than 3.0 degrees, such as less than 2.8 degrees, less than 2.5 degrees, etc. Understood.

A biocompatible surface according to the invention differs from a prior art hydrophobic substrate coated with a hydrophilic layer, in that such a prior art surface has a significantly different contact angle than that of the underlying substrate. In conclusion, the present invention provides a hydrophobic substrate having a predetermined contact angle with essentially the same contact angle for a biologically active portion of the substrate, such as polypeptides, proteins, cells, etc., that is in contact with the substrate. However, it relates to the conversion to a biocompatible material having another function. The biocompatible surface further comprises a second determinant, such as a first determinant capable of bringing a biological cell into reactive contact with the first determinant.
For example, it can include an adhesive polypeptide.

In yet another aspect of the invention, the biocompatible surface is capable of interacting with at least one first determinant (eg, a polypeptide), wherein the first determinant is in an active form. , Preferably in an active conformation. The presence of the first determinant in its functional form and / or active conformation is such that the biocompatible surface comprising the first determinant and, for example, is stable upon contacting and preferably with the first determinant. Improved first determinant between a second determinant, such as a cell, capable of forming a stable bond
Produces determinant-mediated contact.

The first and second determinants, in one embodiment, comprise or consist of cells independently of each other. Cells are preferably fat cells, astrocytes, cardiomyocytes, chondrocytes, endothelial cells, epithelial cells, fibroblasts, ganglion cells, gland cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, It is selected from the group consisting of nerve cells, osteoblasts, pancreatic beta cells, kidney cells, smooth muscle cells, striated muscle cells, and precursors of any of the foregoing. Further, preferred cells are those comprising stromal tissue cells found in loose connective tissue or bone marrow, and preferably endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells, any precursors thereof. .

When the second determinant is a cell, preferably one or more of any of those listed immediately above herein, the first determinant preferably comprises a material according to the invention and a cell of interest. Includes a polypeptide or another biological entity capable of optimizing or stabilizing the bond formed therebetween.

In a first aspect, the invention relates to a material comprising a base layer in contact with a polymer, and further comprising at least one polymer. The material has a first contact angle a, the base layer has a second contact angle b ₀ when not contacted by a polymer, and the contact angle is substantially equal to the contact angle b ₀ .

In one embodiment of this aspect, there is provided a material comprising a base layer in contact with the polymer and further comprising at least one polymer. The material has a first contact angle a, the base layer has a second contact angle b ₀ when not contacted by a polymer, and the base layer is saturated with the polymer as defined herein. _Have another second contact angle b _sat . The relationship between the contact angles is as defined by the ratio R, where R = (b ₀ −a) / (b ₀ −b _sat ) The value of R is between 0 and less than 0.6, including 0.

In another aspect, the invention has a first contact angle and contacts a polymer,
A material comprising a base layer having a second contact angle. The relationship between the first contact angle and the second contact angle is: i) the difference between the second contact angle and the first contact angle in the absence of the polymer, and ii) the difference in the absence of the polymer. A difference between the second contact angle and the substrate contact angle when the substrate is saturated with the polymer as defined herein, as defined by the ratio between: greater than -0.6, greater than 0.6 small.

In another aspect, the invention is directed to a method for contacting with a number of soluble materials having a first contact angle, comprising a polymer, and capable of forming a self-assembled monolayer having a third contact angle, A material comprising a base layer having a second contact angle. The relationship between the contact angles is: i) the third contact angle of the monolayer in the absence of the polymer, and the difference between the first contact angle, and ii) the monolayer in the absence of the polymer. A third contact angle, and the difference between the contact angles of the self-assembled monolayer when the monolayer is saturated with the polymer as defined herein, as defined by: Greater than -0.6 and smaller than 0.6.

All contact angles used for material characterization are advancing contact angles. Pure water is used as a probe liquid, and air saturated with water vapor is used as a probe gas. The material / substrate, pure further double distilled water, and air saturated with water vapor form the three-phase boundary used to measure the contact angle.

The described properties of the biocompatible surface according to the invention, comprising said hydrophobic substrate and said hydrophilic polymer, are such that the first determinant is a functional group conformation or a biologically active form or conformation. To adhere and bond to the surface. The second determinant may remain attached and bound to the functional or active form or conformation, preferably a biologically active form or conformation, by the first determinant and eventually the surface. As such, the properties of the surface, including the underlayer and the polymer and the first determinant, are also useful.

Specific Description of the Invention The following definitions are used in describing the present invention. Defined activity conformation : A protein in a conformation that has the normal biological activity of the host organism. Active form : A protein or biological substance in a form having the same function as when the protein or biological substance is present in the original host or original environment. Adsorption : The removal of molecules from a gas or liquid on the surface of another material, such as a substrate. Advance contact angle : The contact angle as the liquid front advances on the solid material / substrate.

Amphiphilic : A substance containing both a polar water-soluble group and a non-polar water-insoluble group. Sensory cell culture array : A solid or semi-solid support having an orderly structure for attaching sensory cells such as retinal cells. Biocompatible substance : A substance that does not induce an acute or chronic inflammatory response when interacting with a biological substance and does not prevent proper differentiation of the tissue surrounding the transplant. Biological activator : see activator. Biologically active conformation : see active conformation. Biological material : Any substance derived from living organisms, including plants, animals or living parts thereof, such as organs or cells.

Biomaterial : A material that binds to a biological system and evaluates, treats, increases or replaces any tissue, organ or function of the body, for example. Biological ligand : Any ligand of biological origin, including carbohydrates, proteins, or portions thereof, such as oligopeptides, and any combinations and / or derivatives thereof. Biological hybrid organ : A medical device consisting of a combination of biological material and biological material in an active form, such as a specific organ cell. Cell differentiation : The process by which cell precursors become distinct and distinct cell types. Conformational change : A change in the full three-dimensional form of a substance, usually a biological substance. Conformation entropy : The entropy of a polymer, which is determined by the amount of conformation that the polymer can reach.

Conjugate : A plurality of functional molecules joined together chemically. Contact angle (CA) : The angle (θ) represented by the boundary of the liquid phase at the three-phase interface between the solid or semi-solid surface and the liquid and the saturated vapor of the liquid. Various methods are applied to generate the three-phase interface, for example, as a bubble trapping method in which bubbles of saturated vapor of the test solution used are trapped by the test surface. In the context of the claims of the present invention, this is the advancing contact angle characterizing the material surface. Inactivation : To change an active form or active conformation to a less active form or conformation.

Density : mass per volume (concentration) or area (side density). Terminal group : The terminal part of the polymer. Excluded volume : Interaction between segments of a solvated macromolecule or polymer chain that is moving to occupy the same space. Extracellular matrix (ECM) : A network synthesized by cells as the structural and functional support of tissue cells and composed of adhesion proteins such as glycoproteins, laminin, FN, interconnected collagen fibrils, hyaluronate and proteoglycans. Coating : A long and thin sheet of synthetic material. Flexibility : Many conformations can be achieved in contrast to rigidity. Flow rate : The measurement of a certain flow rate per unit time and unit area.

Functionalization : Chemical derivatization that alters structure, properties and / or function. Implantation : Attachment of at least one macromolecule of equal or different molecular units to a substrate via a chemical bond. Head group : a proximal group , that is, a group that forms a bond between a polymer and a base layer. Hydrophilic polymer : Any polymer that has a high surface energy where water droplets spread easily. Hydrophobic polymer : Any polymer with low surface energy that forms prominent water droplets on the surface.

Improving contact : Enhancement of cell attachment and spread upon contact with an abiotic support. Interface : A region or surface that represents the interface between two separate phases in a chemical or physical process. Ionic bond : A bond held together by Coulomb interactions between different charged parts. Latent : exists but is not (yet) active. Lateral monolayer structure : a monolayer formed from a polymer that interacts with neighboring molecules by an inherent exclusion volume, wherein the monolayer is crystalline to form a spontaneously relatively aligned polymer array. It is characterized by a water content of at least 50 percent, not gender.

Layer density : mass per area (2D concentration). Linker : connects two parts or groups or molecules to each other. Macromolecule : any molecule having a molecular weight higher than 400 Da. Membrane : A barrier between the two phases and allows for transport via absorption / diffusion and / or pores. Permeability : A measure of the ability of a membrane to allow transport through the membrane. Light : Here, a physical stimulus to initiate a chemical reaction. Photoreactive polymer : A polymer comprising one or more latent reactive groups. Polymer : A molecule formed by the binding of at least five identical monomers. Pretreatment : addition of functional groups to the substrate.

Reverse contact angle : The contact angle when the liquid front recedes on the solid. Refractive index : the ratio of the phase velocity of electromagnetic radiation in reduced pressure (or atmospheric pressure) to the phase velocity of electromagnetic radiation in a transparent medium. Rigid : essentially inflexible. Saturated Substrate : If the contact angle of the substrate contacted by a plurality of polymers cannot be further reduced by adding more polymers to the surface of the substrate, saturation of the substrate is achieved. More preferably, when the base layer is contacted by a plurality of polymers,
If a significant change in the contact angle cannot be achieved, saturation of the underlayer is achieved.

Self-assembled monolayer : A monolayer formed on a substrate and composed of a self-assembled (stacked or crystallized) component comprises a head group, said head group preferably interacting with the base layer and And the terminal groups are oriented toward a solution. The monolayer is characterized by a crystalline, highly ordered structure and a very low or substantially no water content. Solvation : Molecule or substance is in solution. Synthetic : Any substance that is not of biological origin. Base layer : Any chemical moiety to which a polymer can attach. Surface : the outer part of the object, here the biological material or its precursor.

The present invention teaches a novel method of controlling cell adhesion and the biocompatibility of the associated polymer substrate surface. The novel method comprises the steps of contacting the surface of a hydrophobic substrate, preferably a hydrophobic polymer substrate, with a polymer layer, preferably a monolayer of a flexible polymer, in contact with the surface of the hydrophobic polymer substrate, more preferably a lateral layer. Based on structuring with a monolayer of polymer patterned in

The response of an intrinsic biological system to a design surface according to the present invention is different from the response of such a system to prior art polymers. As well as protein adsorption, antibody binding to adsorbed antigen, fibroblasts, endothelial cells, epidermal cells, hepatocytes and others, ie, biological materials well accepted as a general cell model for tissue-biomaterial interaction. Have been conducted to evaluate the ability of novel biocompatible surfaces to maintain and / or improve the function of the biomaterial to be contacted.

The ability of cells to attach, spread and grow on various surfaces modified according to the present invention is particularly important in that sense. The production of extracellular matrix is one of several important functions of fibroblasts and is generally a property of connective tissue type cells. As a result, the ability of the cells to adhere to the biomaterial according to the present invention is i) within one hour of cell adsorption by the technique of fluorescently labeled FN,
Extracellular matrix formation was studied by microscopic examination.

This study revealed improved cellular functionality as a function of, for example, the molecular weight of the polymer adsorbed on the polymer substrate, and the degree of surface functionalization. The results were measured by typical biocompatibility parameters such as effects on cell adhesion and morphology, focal point formation, extracellular matrix formation, and long-term growth. All of the above are understood to contribute to the improvement in the cell functionality observed as defined above. That is, the results clearly showed that the polymer substrate surface modified according to the present invention had excellent functionality. The raw material, or unmodified polymer, and prior art fully modified surfaces or "characterized by a relatively high degree of surface functionalization"
This functionality is superior when compared to both "coated" surfaces.

The results described herein may be based on the adsorption of the essential biological material, the state of the adsorbent, or the conformation, or the relationship that exists between the bioactive agent and the cell behavior or functionality resulting from the adsorption. Strongly suggests that can be further optimized. Thus, the present invention enables one or more optimal conditions of cell functionality to be determined empirically by theoretical design methods that can be easily controlled by any suitable state of physicochemical surface analysis techniques. . Hence, the response of the adsorbed cells and their biocompatibility can now be predetermined or at least be designed quickly and economically with well-defined and easily adjustable conditions of physicochemical and biotechnological parameters, The present invention achieves that objective by significantly improving the state of the art methods of providing biomaterials.

The materials that can be processed according to the invention have at least, for example, transparency, refractive index, electrical conductivity, thermal stability, resistance to hydrolysis, or at least, even if their physicochemical properties are not excellent for any given application. Membrane formation (eg, from cell culture dish to membrane)
But have poor, if not completely useless, cell attachment, growth and functionality due to their undesirable physicochemical surface properties. Things. The surface structure of the biomaterial treated in accordance with the present invention may be a stochastic or predetermined or controlled permeability (potentially constructed as a temporary or permanent cell support, described immediately below). For example, it may be a porous structure with micro or macro porous flat sheets or hollow fiber membranes).

The two-step modification method disclosed herein (see FIG. 4) preferably produces covalent, patterned monolayers. The structure and structure or functionality of this layer may be designed or predetermined in the first adsorption stage by the synthesis of the polymeric conjugate and then optionally according to a given set of environments. By covalent implantation, stable attachment (ie, implantation) to the underlying polymeric material (base polymer) is easily achieved. For example, management of "design parameters" such as the molecular structure of the amphiphilic polymer and the concentration and / or solubility of the polymer can be left to those skilled in the art of manufacturing complex polymers.

The molecular weight and / or size of the amphiphile determines the molar density (ie, macromolecules per surface area), at least to some extent. Increased interaction between the amphiphilic polymer and the polymer base layer tends to increase the layer density. Similarly, a high concentration of the amphiphilic polymer in the first stage (see FIG. 4) or a decrease in the solvency of said amphiphilic polymer also contributes to an increase in the layer density. It is believed that the change in solubility can be achieved, for example, by salt concentration, pH, temperature or the polarity of the solvent. Application of the amphiphile by spray coating and subsequent drying followed by ultraviolet (UV) or visible (Vis) irradiation may be an alternative technique. Those skilled in the art are familiar with the physicochemistry of polymers and macromolecules required to change layer density.

When the biomaterial is a membrane, the polymer substrate is substantially impermeable to water, while when the biocompatible material is a membrane, the polymer substrate is porous. The lateral layers made according to the present invention are characterized by the amphiphilic and amphiphilic-amphiphilic intermolecular and intramolecular interactions of the polymer. The strong repulsion between amphiphiles due to the inherently large excluded volume leads to individual adsorbed molecules that can form "self-assembled" structures on the sides. According to one preferred embodiment of the invention, the surface functionalization is mediated by a conjugate of a photoreactive hydrophilic flexible polymer comprising a well-defined building block modular composition. . In one particularly preferred embodiment, the module composition comprises (potential reactive head group)-(leading group)-(subject)-(functional end group).

The present invention seeks to provide a substrate surface having desirable physical properties and comprises a plurality of steps of contacting the substrate with a composition comprising a plurality of polymers having desirable physical properties. Each macromolecule consists of a potentially reactive head group covalently linked to the subject, optionally via a linker group, and optionally also a leader group and a functional end group. The latent reactive head group can provide one or more active species, such as free radicals, in response to an external stimulus, and covalently attach the polymer to the substrate through the residues of the latent reactive head group.

Although the macromolecules are spatially oriented so that one or more potential reactive groups can be covalently bonded in close proximity to the substrate surface, the method according to the present invention provides a method for applying latent stimuli by applying an external stimulus. Further activating the reactive group to conjugate the polymer to the substrate. The external stimulus used is preferably electromagnetic radiation, since the layer structure established by "self-assembly" is not disturbed by this type of radiation and the polymer substrate remains at least substantially intact, More preferably, the radiation is in the ultraviolet, visible or infrared region of the electromagnetic spectrum.

The degree of conversion can be selected, for example, by the amount of UV / Vis donated, and generally 100% conversion is attempted. The response to the active step of the method can be tuned by choosing various potentially reactive groups. The reactivity of the photochemically generated reactive species can be selected according to the polymer base layer structure. Thus, for example, aryl nitrenes from aryl azides are, after UV / Vis excitation, all polymers having -NH, -OH or -CH groups and aromatic ketones via an insertion reaction. It is well-known that hydrogen abstraction leads to an insertion reaction with all polymers having at least a —CH group.

[0087] The potentially reactive head group of the macromolecule utilized in the present invention comprises one or more covalently linked potentially reactive groups. As defined herein, a potentially reactive group is a group that undergoes the generation of an active species that is covalently attached to an adjacent carrier surface in response to a particular applied external stimulus. A latent reactive group is a group of atoms in a molecule that, when conserved, retains its covalent bond unchanged but forms a covalent bond with another molecule when activated. Latent reactive groups give rise to active species such as free radicals, nitrenes, carbenes and excited state ketones due to absorption of external electromagnetic or kinetic (thermal) energy.

The potential reactive groups are selected to be sensitive to various parts of the electromagnetic spectrum,
Potentially reactive groups that react in the ultraviolet, visible or infrared portion of the spectrum are preferred. Azides constitute a preferred class of potentially reactive groups, including phenyl azide,
Allyl azides such as -azidobenzoic acid and 4-fluoro-3-nitrophenyl azide, acyl azides such as benzoyl azide and p-methylbenzoyl azide,
Ethyl azidoformate, azide formate such as phenyl azidoformate, sulfonyl azide such as benzenesulfonyl azide, and phosphoryl azide such as diphenylphosphoryl azide and diethylphosphoryl azide are included. Diazo compounds constitute another class of latent reactive groups, diazomethane or a diazoalkane, such as diphenyl diazomethane (-CHN ₂₎ and as diazo acetophenone and 1-trifluoromethyl-1-diazo-2-pentanone Diazoketones, diazoacetic acid t-
Butyl, diazoacetates such as phenyl diazoacetate and beta-keto-alpha-diazoacetates such as t-butyl alphadiazoacetoacetate.

Other potentially reactive groups include aliphatic azo compounds such as azobisisobutyronitrile, diazines such as 3-trifluoromethyl-3-phenyldiazirine, ketene and diphenylketene. Ketones (-CH = C = O) and photoactivatable ketones such as benzophenone and acetophenone. Peroxide compounds are considered as another class of potentially reactive groups, including dialkyl peroxides such as di-t-butyl peroxide and dicyclohexyl peroxide and diacyl peroxides such as dibenzoyl peroxide and diacetyl peroxide and ethyl peroxybenzoate. Such peroxyesters are included.

Activation of the latent reactive group that results in the formation of a covalent bond with the surface to which the macromolecule is attached causes the macromolecule to be covalently bonded to the surface by the residue of the latent reactive group. As can be seen from the foregoing disclosure, photoreactive groups are generally aromatic, and therefore generally hydrophobic rather than hydrophilic. When a relatively hydrophobic reactive head group, such as an aromatic photoreactive group, is present, the macromolecule aligns itself with the surface of the hydrophobic substrate in aqueous solution, so the relatively hydrophobic reactive head group Is preferentially carried near the surface of the carrier, while the rest of the macromolecule, the major part and the functional end groups, point away from the surface of the hydrophobic substrate. This feature allows the macromolecule to be tightly covalently bonded to the relatively hydrophobic carrier surface, thus contributing to the formation of a biocompatible substrate surface as defined above.

According to the above, the amphipathic properties and thereby the orientation and the achieved grafting concentration of macromolecules on the substrate surface can be increased by incorporating hydrophobic leading groups into the macromolecules. The leader group is a bifunctional group, preferably located by a linker group, between the potentially reactive head group and the rest of the macromolecule, i.e. between the main part and the functional end groups. The leading group is intended to increase the hydrophilic properties of the macromolecule in order to generate a favorable orientation enhancement of the macromolecule's potentially reactive head group to generate bonding at the substrate surface and to increase the achieved grafting concentration It is hydrophobic from the above. Preferred types of leader groups are aliphatic, linear or slightly branched or cycloaliphatic groups, all of which preferably have 6 to 18 carbon atoms or a combination thereof, simultaneously with a mono- or polycyclic aromatic group or a combination thereof. And combinations of the above aliphatic groups.

The major portion of the macromolecule is preferably hydrophilic, uncoiled in an aqueous environment and exhibits an excluded volume. It can be a natural or synthetic polymer. Such polymers include oligomers obtained by addition or condensation polymerization, homopolymers and copolymers and parts thereof such as oligosaccharides, polysaccharides, oligosaccharides and polypeptides or extended oligopeptides, and the like. Of natural polymers.

The macromolecules making up the main part comprise several different macromolecular types prepared by terminal or side chain grafting, including hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, cellulose acetate and cellulose butyrate. Such cellulose-based products, hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate,
Acrylic resin such as acrylic acid, methacrylic acid, acrylic resin polymerized from acrylamide and methacrylamide, vinyl resin such as polyvinylpyrrolidone and polyvinyl alcohol, polycaprolactam, polylauryl lactam, polyhexamethylene adipamide and polyhexamethylene dodecanediamide Nylons, polyurethanes, polylactic acid, amylose, dextran, linear polysaccharides such as chitosan and hyaluronic acid, amylopectin, and branched polysaccharides such as hyaluronic acid and hemicellulose.

The macromolecule itself is preferably at least about 500 Da, most preferably about 10,000 Da.
, Is strongly hydrophilic and dissolves in water at 25 ° C. to at least about 0.5% by weight. In a preferred embodiment, the main portion, such as ethoxy (-CH ₂ -CH ₂ -O-) or isopropoxy _{(-CH 2 -CH (CH 3)} -O-) comprises repeating units of such groups, among which PEG is most preferred. The functional end group comprises all biological or synthetic molecules or chemical moieties that can permanently or reversibly bind cells, viruses and the like to the surface via the macromolecular main part, It has a function of charging or chelating together with a hydroxy, amino, carboxyl, sulfonic acid, activated ester or epoxy group.

In addition, the functional end groups can be selected from many types of compounds or fragments thereof that confer to the modifying substrate a general or specific “biophile” as defined below. Generally, a biophilic functional terminal group includes, for example, whole cells, fractionated cells,
It promotes the binding, attachment or adsorption of biological substances such as cellular organelles, proteins, lipids, polysaccharides, simple carbohydrates, complex carbohydrates and / or nucleic acids.
Commonly bio Fi Rick functional end groups, COO ^-, -PO ₃ H ^- or 2-imidazolo hydrophobic group or an alkyl group having a such a charged part of the group, and extracellular matrix proteins, FN, collagen, laminin, serum Compounds and fragments of compounds such as albumin, polygalactose, sialic acid and various lectin-binding sugars are included.

Specifically, a biophilic functional end group is one that selectively or preferentially binds, attaches, or adsorbs a specific type or type of a biological substance to identify or isolate a specific material from a material mixture, for example. is there. Specific biophilic materials include antibodies or antibody fragments and their antigens, cell surface receptors and their ligands, nucleic acid sequences, and many others known to those of ordinary skill in the art. The selection of an appropriate biophilic end group depends on the biological material to be coupled, the required binding affinity, availability, ease of equipment and cost. Such choices are within the knowledge, abilities, and judgment of the normal party.

In preparing biodegradable coatings or coatings that degrade under preset environmental conditions, it is desirable to incorporate into the macromolecule a moiety that causes enzymatic or chemical hydrolysis of the coating. Suitable components include amino acids such as alanine, valine, leucine, proline, methionine, aspartic acid, threonine, serine, glutamic acid, glycine, cysteine, phenylalanine, lysine, histidine, arginine and aminobutyric acid. As an alternative, ester bonds which are unstable in hydrolysis can likewise be applied. All of these moieties are representative of the linker group, if present, but are also incorporated into the major or leading group of the macromolecule.

The lateral density of a monolayer of macromolecules in the present invention can be determined, for example, by i) changing the amount and / or concentration of macromolecules in solution during “self-assembly”, or ii) using a mixture of macromolecules. Macromolecules can be adjusted, for example, by variations in different molecular weights or other structural features of the macromolecules (eg, branched versus unbranched), or iii) appropriate selection of solution conditions in the adsorption application of the macromolecules, eg, dissolution It can be adjusted by selecting force, ionic strength, temperature or pH. The step of photochemical grafting does not disturb this "self-assembly" mode, nor does it cause any substantial degradation of the underlying surface of the polymeric substrate.

The base layer may be a tactile surface of the film or film, or a surface of a contact lens or surgical implant, or a surface provided by small particles or the like in an emulsion or other suspension, or a powder or soft gel. Comprising a surface such as a surface. The present invention has the unique advantage of providing a method for providing a simple, fast, and covalently attached macromolecular coating to a non-pretreated, definable surface (eg, a solid) in a simple, rapid, and economical manner. I have it.

[0100] Preferred embodiments of the present invention are described below. The materials of the present invention include soluble materials in the form of molecules capable of forming a self-assembled monolayer. Also, the base layer has preferably been subjected to a pretreatment or modification that is the result of contacting the base layer with a charged group or a hydrophilic compound and / or binding in a usable state. As is apparent from the above, the contact angle of the material is a advancing contact angle.
t angle). In one embodiment, the advancing contact angle is in the range of 50 to 140 degrees, preferably in the range of 60 to 125 degrees, e.g. in the range of 70 to 120 degrees, e.g. in the range of 75 to 110 degrees, e.g. Range from 80 degrees to 100 degrees.

However, one material also shows a receding contact angle, in which case the contact angle is in the range of 30 to 120 degrees, preferably in the range of 40 to 110 degrees,
For example, a range of 50 to 100 degrees, for example, a range of 60 to 90 degrees, for example, 70 to 8
The range is 0 degrees. The difference between the second contact angle and the first contact angle when no macromolecule is present, and the ratio of the difference between the second contact angle and the base layer contact angle when no macromolecule is present, If the underlayer is saturated with the macromolecule as defined herein-
Greater than 0.6 and less than 0.6, and preferably greater than 0 and less than 0.50, for example 0.40
Less than, for example, less than 0.30, in the example, less than 0.25, such as less than 0.20, in the example, less than 0.15, such as less than 0.10, in the example, less than 0.05.

When the contact angle is a receding contact angle, the ratio is preferably less than 0.40. The difference between the third contact angle of the monolayer and the first contact angle in the absence of macromolecules, and the third contact angle of the monolayer and the contact angle of the self-assembled monolayer in the absence of macromolecules Is greater than -0.6 and less than 0.6 if the base layer is saturated with the macromolecule as defined herein, preferably greater than 0 and less than 0.50, for example less than 0.40. Small, for example less than 0.30, in the example less than 0.25, for example less than 0.20, in the example less than 0.15, for example less than 0.10, in the example less than 0.05.

In a detailed and preferred embodiment, contacting with a first determinant comprising a compound selected from the group comprising a polypeptide, or a portion thereof, a portion of a nucleic acid, a portion of a carbohydrate and a portion of a lipid and combinations thereof. A material that is capable of maintaining the compound in a biologically active form. More preferably, the compound is a polypeptide or a part thereof.

A material is provided that further comprises the first determinant comprising the compound, wherein the first determinant maintains a biologically active form when contacting the substrate and / or the macromolecule. I do. The biologically active form preferably refers to the biologically active conformation. The bioactive form or conformation is preferably maintained and / or improved and / or stabilized by the cooperation of the substratum and the macromolecule. The bioactive form or conformation is preferably maintained and / or improved and / or stabilized by contacting the substrate with the macromolecule. The material of the present invention is preferably biocompatible.

In the present invention, the weight gain per area unit due to the portion of macromolecules consisting essentially of PEG or poly (ethylene oxide) (PEO) is square nanometers (nm ² ).
^Less than 2.0 × 10 ⁻²² grams (g) per square nanometer (nm ² ), for example less than 1.0 × 10 ⁻²² grams (g) per square nanometer (nm ² ), for example 0.8 per square nanometer (nm ² )
In × 10 ^-22 grams less than, provides square nanometer (nm ²⁾ Less than 0.5 × 10 ^-22 grams per, for example, less than square nanometer (nm ²⁾ 0.3 × 10 ^-22 grams per Material Examples I do.

A material is also provided in which the base layer is in contact with a plurality of soluble compounds capable of forming a layer of self-assembled macromolecules, preferably n-alkane chains having 8 to 24 carbons. The macromolecules of the invention are characterized by an excluded volume. The base layer preferably comprises a hydrophobic polymer, and in one embodiment the base layer is at least essentially flexible and / or a coating. However, the substrate may at the same time be essentially rigid or at least essentially non-flexible. in this case,
The base layer comprises a crystalline structure capable of supporting a self-assembled monolayer such as gold, silicon oxide and similar crystalline structures, and / or the structure is smooth on a nanometer scale.

The macromolecules of the present invention comprise a hydrophilic polymer or an amphiphilic polymer. Macromolecules preferably have a molecular weight of more than 400 Da, for example a molecular weight of more than 1,000 Da, for example a molecular weight of more than 5,000 kDa, for example a molecular weight of more than 10,000 Da, for example
It has a molecular weight of more than 50,000 Da, for example having a molecular weight of more than 100,000 Da. The macromolecules of the invention are preferably conjugates comprising a head group, a leader group, a linker group, a polymer chain or a main part and a functional end group. The head group can form a chemical bond (see FIG. 5), such as an ionic bond (see FIG. 6),
Adsorb to or into the substrate (see FIG. 7) or become entangled with the substrate (see FIG. 8). The headgroup can form a self-assembled monolayer (see FIG. 9).

Preferred leader groups are bifunctional groups containing linear or slightly branched aliphatic groups. Leading groups can also form and / or stabilize a self-assembled monolayer. Preferred linker groups are capable of being enzymatically or chemically hydrolyzed,
It is unstable to hydrolysis but is essentially stable to fission in practical environments. The polymer chain or major portion is preferably hydrophilic and unfolded in an aqueous environment to show excluded volume. Functional end groups can be permanently or reversibly associated with other biological or synthetic molecules and materials.

The first determinant as defined herein comprises a biologically active compound containing a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion and a lipid portion and any combination thereof. The biologically active compound is preferably selected from the group consisting of polypeptides of the membrane associated and / or extracellular matrix produced by microbial, plant or mammalian cells. In other embodiments, the biologically active compound is a polypeptide, antibody, polyclonal antibody, monoclonal antibody, immunodeterminant, antigenic determinant, receptor, receptor binding protein, interleukin, cytokine, cell differentiation factor, cell growth factor and receptor. It is selected from the group consisting of body antagonists.

The biologically active compound is also a synthetic polypeptide or part thereof capable of contacting said substratum and / or said macromolecule. Preferably, the biologically active compound is an adhesion polypeptide, preferably FN or vitronectin. The biologically active compound is preferably between the material and a biological entity such as a biological cell or a virus or a part thereof containing a polypeptide or a part thereof, a nucleic acid part, a carbohydrate part and a lipid part and any combination thereof. Causes improved contact. In a particularly preferred embodiment, the material of the present invention further comprises a second determinant as defined herein. A second determinant is a biological cell or virus comprising a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion and a lipid portion and any combination thereof;
Or a biological entity such as a part thereof.

Biological entities are also preferably polypeptides, antibodies, polyclonal antibodies, monoclonal antibodies, immunodeterminants, antigenic determinants, receptors, receptor binding proteins, interleukins, cytokines, differentiation factors, growth factors and receptors. Selected from the group consisting of antagonists to The biological cells or parts thereof are preferably microbial cells, including mammalian cells such as human cells and animal cells, plant cells, eukaryotic microbial cells such as yeasts and fungi, and prokaryotic microbial cells such as bacteria.

The second determinant is also a microbial virus, including mammalian viruses such as human and animal viruses, plant viruses, eukaryotic microbial viruses such as yeast and fungal viruses, and prokaryotic microbial viruses such as bacteriophages. . In one embodiment, the substrate is porous, and is preferably a membrane. The flow of water through the material is preferably substantially unchanged compared to the flow of water through the porous substrate. In other embodiments, the substrate is non-porous and / or substantially impermeable to water.

A method for controlling cell growth and / or cell proliferation and / or cell differentiation in vitro,
Alternatively, a material used in a method of separating and / or isolating a biological substance in vitro or a method of preparing a biohybrid organ in vitro is provided. In another aspect, there is provided a material for use in a method of diagnosing a human or animal body, a method of treating a human or animal body, or a method of performing surgery on a human or animal body.

Provided are a material used in a method for producing a biohybrid organ in a living body and a material used as a carrier when the drug is transported in vivo to a human or animal body in need of the drug. In another aspect, a material used in a method of controlling cell growth and / or cell proliferation and / or cell differentiation in a living body, and a material used in a method of separating and / or isolating a biological component in a living body are provided. provide. In another aspect, there is provided a composition comprising a material of the invention and a physiologically acceptable carrier. The invention also relates to a pharmaceutical composition comprising a material of the invention or a composition or pharmaceutically active ingredient as defined herein and optionally a pharmaceutically active carrier.

The pharmaceutically active compounds are preferably enzymes, hormones, cytokines, colony stimulating factors, vaccine antigens, antibodies, clotting factors, regulatory proteins, transcription factors, receptors,
Structural protein, angiogenic factor, human growth hormone, factor 8, factor 9, erythropoietin, insulin, alpha-1, antitrypsin, calcitonin,
Glucocerebrosidase, low density lipolipid (LDL) receptor, IL-2 receptor, globin, immunoglobulin, catalytic antibody, interleukins, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH) , Leptin, interferons,
Nerve growth factors, basic fibroblast growth factor (bFGF), transforming growth factor (TGF
), Transforming growth factor beta (TGF-beta), acidic FGF, epidermal growth factor (EGF)
, Endothelial cell growth factor, platelet-derived growth factor (PDGF), transforming growth factors, endothelial cell stimulating angiogenic factor (ESAF), angiogenin, tissue prosminogen activator (t-PA), granulocyte colony stimulation Factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF).

Use of the material or the composition or the pharmaceutical composition of the present invention in a method of treatment performed in the human or animal body, a method of surgery performed in the human or animal body, or a method of diagnosis performed in the human or animal body. provide. In other embodiments, the material or the composition or the pharmaceutical composition is used to deliver a biohybrid organ in vivo or to deliver the drug to a human or animal body in need of the drug in vivo. It is provided for use as a carrier.

The present invention also provides for the use of the material or the composition or the pharmaceutical composition in a method of controlling cell growth and / or cell proliferation and / or cell differentiation in vivo, the use of the material in vivo. Use in a method of separating and / or isolating a biological component, or use of the material in a method of cell growth and / or cell proliferation and / or cell differentiation in vitro, or preparation of a hybrid organ in vitro of the material And the use of the material in the manufacture of implantable organs or parts thereof. The material of the present invention can be used as a carrier for a pharmaceutical active ingredient or a pharmaceutical composition.

Further, a method for controlling cell growth and / or cell proliferation and / or cell differentiation in vitro, comprising contacting a cell with the material or the composition of the present invention or the drug composition of the present invention; A method is provided for culturing the cell and the material under conditions that allow the cell to grow and / or proliferate and / or differentiate. The present invention also provides an ex vivo separation and / or isolation of a biological material comprising the step of contacting said biological material to be separated and / or isolated with said material or said composition or said pharmaceutical composition of the present invention. And a method of culturing the biological material and the material under conditions for separating and / or isolating the material.

A method for producing a biophilic organ in vitro, the method comprising contacting a biohybrid organ cell with the material or the composition or the pharmaceutical composition of the present invention and the biohybrid organ. A method is provided for culturing the biohybrid organ under conditions to be produced. The invention likewise relates to the following method, which is a particularly preferred embodiment: A method of treatment performed on the human or animal body, said method comprising the step of contacting said body with a material or composition or pharmaceutical composition of the invention. including. A method of surgery performed on the human or animal body, said method comprising the step of contacting said body with a material or composition or pharmaceutical composition of the present invention.

A diagnostic method performed on the human or animal body, said method comprising the step of contacting said body with a material or composition or a pharmaceutical composition of the invention and resulting directly or indirectly from said material. Detecting the signal. A method for controlling cell growth and / or cell proliferation and / or cell differentiation in a living body, the method comprising: contacting a cell with a material or a composition or a drug composition of the present invention; Culturing under conditions where the cells grow and / or proliferate and / or differentiate.

A method for separating and / or isolating a biological substance in a living body, wherein the method comprises separating and / or isolating a biological substance.
Or contacting the biological material to be isolated with the material or composition or the pharmaceutical composition of the present invention, and culturing the biological material and the material under conditions for separation and / or isolation. A method for producing a biohybrid organ in a living body, the method comprising: contacting a biohybrid organ cell with a material or composition or a pharmaceutical composition of the present invention; Culturing the cells.

A method of delivering a drug in vivo to a human or animal body in need of the drug, the method comprising: contacting the body with the pharmaceutical composition of the present invention; and conditions for delivering the drug. Culturing said body and said drug composition below. Further preferred embodiments of the present invention are exemplified herein below. US Pat. No. 5,201,715, incorporated herein by reference, relates to a target object having a characteristic ultrasonic signature for subcutaneous implantation. By placing the target within the implantation site, the target can be distinguished from surrounding tissue by ultrasonic acoustic measurements. The object under the skin can be located with non-invasive ultrasound acoustic measurements. The signature comprises the reflection of ultrasonic waves from the object.

Accordingly, one aspect of the present invention is directed to a device for implantation under the skin that, when implanted, allows positioning under the skin by non-invasive ultrasound. The device includes a target comprising the biocompatible material of the present invention including at least one and preferably a plurality of ultrasound reflecting surfaces, wherein the at least one or more of the ultrasound reflecting surfaces are included. The combination provides a characteristic ultrasonic acoustic sounding signature. The biocompatible material preferably has an acoustic velocity different from the acoustic velocity of human tissue, and the object optionally further comprises a laminated structure comprising a substantially planar layer laminated with the biocompatible material of the present invention. . The object preferably has a single structure.

US Pat. No. 5,976,780, which is hereby incorporated by reference herein, relates to a device for macroencapsulation for somatic cells. Accordingly, the present invention, in one aspect, relates to an implanted or implanted device, comprising: i) from the material of the present invention having porous and terminal and fibrous walls that allow the selective passage of nutrients, gaseous substances and metabolites and only the passage of substances with a molecular weight of less than about 30,000 daltons; And ii) a mixture of living cells, preferably somatic and mammalian cells, and an alginate gel suspended within said fibers.

Similarly, the present invention provides a device which does not have large voids in the wall surface, but has a porosity sufficient to prevent the passage of donor antigens and cytokines through the wall surface. The device preferably comprises a fiber of the material of the present invention capable of inhibiting complement activation. Said somatic cells are preferably selected from the group consisting of neurons, endocrine and hepatocytes, said cells preferably being free of passenger leukocytes.

Also provided are implantable and implantable devices, but the device comprises: i) a hollow fiber having a terminal and fibrous wall with porosity to selectively allow nutrient, gaseous and metabolites to selectively pass and comprising the material of the invention; ii) viable cells, preferably Is a viable mixture of somatic and mammalian cells and alginate gels suspended in said fibers. The alginate optionally comprises ultrapure alginic acid substantially free of divalent metal poisons, (i) the endotoxin content is preferably less than 750 EU / g, (ii
) The protein content preferably comprises less than 0.2% and (iii) the content of G monomers, dimers and trimers preferably comprises more than 60%.

US Pat. No. 4,624,669, which is incorporated herein by reference, relates to a corneal inlay for endocorneal transplantation, comprising a material such as polysulfone, wherein the nutrients and fluids pass from the lower layer of the cornea to the outermost layer of the cornea. Includes multiple holes to facilitate movement to Thus, in one embodiment of the present invention relates to an inlay, wherein the inlay comprises: i) an optical lens comprising the material for endokeratin implantation of the present invention; and ii) a plurality of holes 0.001 mm to 0.1 mm in diameter, a bottom surface. From the outermost surface to allow nutrients to pass through the cornea.

Similarly, the corneal inlay comprises: i) an optical lens comprising the material for endokeratous implantation of the present invention; and ii) a plurality of slits having a maximum width of 0.01-0.05 mm and a maximum length of 0.05-0.05. With 1.0 mm, the slit extends from the bottom surface to the outermost surface and is for passage through the cornea. U.S. Pat. No. 5,213,721, which is incorporated herein by reference, relates to a porous device comprising a plurality of holes aligned in a predetermined geometry. Such holes are obtained in the process of repeated drawing. Prior to the first drawing operation, each hole is filled with a material that can be dissolved with some kind of reagent and drawn with the basic material. The extent of the drawing provides a porous device containing holes that significantly reduce the cross-sectional area.

Accordingly, there is provided a device comprising a material of the present invention for use in any of a skeleton, a contact lens, an intrakeratinous inlay, an intraocular lens, a medical filter or a similar structure with a stoma. Accordingly, there is provided an optical device such as a skeleton or contact lens comprising the material of the present invention. U.S. Pat.No. 5,965,125, incorporated herein by reference, relates to an implantable device having a body of matrix material made of insoluble collagen fibers,
In it, i) a plurality of vertebrate cells, and ii) a plurality of fine particles including fine particles mainly composed of polysulfone are arranged.

Thus, in one embodiment of the invention, a composition comprising a matrix material, preferably a matrix material comprising insoluble collagen fibers, embedded in the main part of said matrix material comprises: i) cultured cells A plurality of, preferably vertebrate cells, more preferably a plurality of genetically engineered vertebrate cells, wherein said cells are capable of expressing a medically useful bioactive compound comprising a polypeptide, and ii) at least said microparticles Is a plurality of fine particles containing the material of the present invention.

Cultured vertebrate cells are preferably adipocytes, astrocytes, chondrocytes, cardiomyocytes, endothelial cells, epithelial cells, fibroblasts, ganglion cells, gland cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes , Myogenic cells, neurons, osteoblasts, pancreatic beta cells,
It is selected from the group consisting of kidney cells, smooth muscle cells, striated muscle cells and precursors of any of the above.

The cultured vertebrate cells are preferably transfected cells, preferably transfected human cells containing exogenous DNA encoding a medically useful biologically active compound comprising a polypeptide. Cultured vertebrate cells are preferably transfected cells containing exogenous DNA containing a regulatory sequence that activates the expression of a gene encoding a polypeptide with the medically useful biologically active compound, preferably the gene, It is endogenous to the vertebrate cell before or after transfection.

The biologically active compounds, preferably polypeptides, preferably enzymes, hormones, cytokines, colony stimulating factors, vaccine antigens, antibodies, clotting factors, regulatory proteins, transcription factors, receptors, structural proteins, angiogenesis Factor, human growth hormone, factor VIII, ninth factor, erythropoietin, insulin, alpha-1, antitrypsin, calcitonin, glucocerebrosidase, low density lipolipid (LD
L) receptor, IL-2 receptor, globin, immunoglobulin, catalytic antibody, interleukins, insulin-like growth factor 1 (IGF-1), parathyroid hormone (PTH),
Leptin, interferons, nerve growth factors, basic fibroblast growth factor (
bFGF), transforming growth factor (TGF), transforming growth factor beta (TGF-beta),
Acidic FGF, epidermal growth factor (EGF), endothelial cell growth factor, platelet-derived growth factor (PDGF
), Transforming growth factors, endothelial cell stimulating angiogenic factor (ESAF), angiogenin, tissue prosminogen activator (t-PA), granulocyte colony stimulating factor (G-
CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF).

In a preferred embodiment, a biologically active compound, preferably a medically useful polypeptide, is administered to a patient whose blood has been partially shunted such that the polypeptide is secreted by the cells in a hybrid matrix with blood. To In general, any suitable method known to the person skilled in the art can be used to adjust the matrix of the invention and can be applied. For example, short circuiting blood to a device containing a perm-selective surrounding a matrix containing the material of the present invention will deliver a therapeutic product of the matrix to the blood. Artificial pancreas (Sull
Devices similar to ivan et al., Science 252: 718-721, 1991) can be used for this purpose.

In another preferred embodiment, a hybrid matrix comprising a material of the invention is one means of producing a polypeptide in vitro. The method includes the steps of: placing a hybrid matrix containing the material of the present invention under conditions in which the cells express and secrete a polypeptide of interest in the matrix; contacting the matrix with a predetermined liquid to allow the cells to Secreting the polypeptide into the liquid; for example, obtaining the polypeptide from the liquid using standard purification techniques appropriate for the resulting polypeptide.

In a preferred embodiment, the matrix comprising the material of the invention is immobilized on a surface and immersed in the liquid; alternatively, the matrix is freely suspended in the liquid. Cells embedded in a hybrid matrix function favorably in a relatively confined space. Furthermore, the first step of the purification, for example, of the expressed polypeptide (removing the cells from the medium) can be performed considerably more efficiently with the matrix according to the invention than with the most standard methods of cell culture. U.S. Pat.No. 5,679,924, incorporated herein by reference, encloses tumor cells in a semipermeable membrane hollow fiber compartment, implants the encapsulated fiber compartment into the mammal, then treats the mammal with cancer therapy, and then treats the hollow The present invention relates to a method for evaluating the effect of cancer treatment on cells in a fiber compartment and measuring the effect of cancer treatment.

Accordingly, one embodiment of the present invention relates to a method for measuring the effect of a cancer treatment, the method comprising the steps of: i) comprising a material of the present invention and having a pore size capable of passing nutrients, but wherein Providing an elongated area of translucent hollow fibers having said pore size that excludes components capable of inducing tissue rejection with components of the immune system of ii) injecting said tumor cells into said hollow fibers; The ends of the hollow fibers are closed, and iii) the sealed hollow fibers are implanted intraperitoneally or subcutaneously in a non-human mammal. iv) perform cancer treatment and v) observe the effect of the treatment on cells in the implanted hollow fibers. The mammal is preferably an immunocompetent eg rat, and the tumor cells are preferably leukemia cells, preferably autologous leukemia or tumor cells obtained from a patient's tumor. Tumor cells are in the form of a suspension or tissue explant.

US Pat. No. 5,830,708, incorporated herein by reference, relates to a method for producing a naturally secreted human extracellular matrix material and a composition containing the extracellular matrix material. The method involves culturing human cells secreting extracellular matrix in vitro on a biocompatible three-dimensional structure. Accordingly, one aspect of the present invention relates to a method for producing a human, naturally secreted extracellular matrix material, said method comprising the steps of: i) a) living tissue, optionally in vitro Culturing living tissue, preferably comprising human stromal cells such as fibroblasts, in the tissue prepared in b), b)
Providing a connective tissue protein naturally secreted by living tissue, said connective tissue being attached to and substantially containing the material of the invention; ii) killing cells in the living tissue; And iii) removing the killed cells and any cellular content from the material of the invention, and iv) collecting the extracellular matrix material deposited on the structure.

The method optionally further comprises homogenizing the collected extracellular matrix material, treating the bridge, or suspending the extracellular matrix material in a physiologically acceptable carrier. Said stromal cells, preferably fibroblasts, of living stromal tissue may be loose connective tissue or bone marrow and preferably endothelial cells, pericytes, macrophages, monocytes,
Cells found in leukocytes, plasma cells, mast cells or fat cells.

In one aspect of this aspect of the invention, an injectable material for augmenting soft tissue and related methods of use and a method of manufacture that overcomes the shortcomings of the state of the art, such as bovine injectable collagen and similar injectable materials. I will provide a. Injectable materials of the invention comprise preparations derived from naturally secreted extracellular matrix as well as naturally secreted extracellular matrix preparations. These preparations are biocompatible, biodegradable and can promote connective tissue deposition, angiogenesis, re-epithelialization and fibrosis, which are useful in repairing skin and other tissue defects. These extracellular matrix preparations are used in repairing tissue defects by injection at the site of the defect.

In another embodiment of the invention, the preparation can be used in a highly modified system for in vitro tissue culture. A naturally secreted extracellular matrix coated on a three-dimensional structure containing the material of the present invention is used for cultured cells that need to adhere to a carrier to grow but do not adhere to a conventional tissue culture dish be able to.
In addition to culturing cells in the coated structure, the extracellular matrix secreted by the cells into the structure can be collected and used in coated dishes for tissue culture. The extracellular matrix, which acts as a base substrate, allows cells that would not normally adhere to a conventional tissue cell dish base substrate to adhere to and subsequently grow.

Further, another aspect of the present invention relates to a novel method for measuring the ability of a specific cell to chemotaxis. The method involves implanting the cell type in question at one end of a native extracellular matrix-coated three-dimensional structure containing a material of the invention and measuring the cross-sectional distance of the structure by the cell over time. It is an advantage that an equivalent amount of extracellular matrix is found in the body in vitro, since the extracellular matrix is naturally secreted by the cells onto the structure. Such assays may indicate, for example, whether the isolated tumor cells have metastasized or whether certain immune cells have been able to migrate across or have chemotacticized the structure. It suggests that it has a high cell chemotaxis ability.

In another aspect of the present invention, there is provided a method of producing a material of the present invention, the method comprising providing a substrate having a second contact angle, and comprising a plurality of macromolecules. Contacting with the composition. The method preferably involves the production of a material as described above. The substrate preferably comprises a hydrophobic polymer and the substrate is subjected to a pretreatment before contacting the macromolecules. Pretreatment is effective in increasing the wettability of the base layer.

The macromolecules of the present method comprise a hydrophilic polymer, preferably a potentially reactive polymer. The macromolecule preferably has a molecular weight (MW) of more than 400 Da. The macromolecule comprises a conjugate containing preferred head groups, linker groups, polymer chains and functional end groups. The head group is preferably a photoreactive allyl azide head group. The macromolecule optionally includes a modifier, preferably a modifier that contacts the substrate and forms a self-assembled monolayer.

According to the method of producing a material of the invention, said method comprises the further step of contacting said material with a first determinant comprising a biologically active compound. Biologically active compounds preferably include polypeptides, antibodies, polyclonal antibodies, monoclonal antibodies, immunodeterminants, antigenic determinants, receptors, receptor binding proteins, interleukins, cytokines, cell differentiation factors, cell growth factors or receptors. Antagonist. Biologically active compounds are membrane-associated and / or extracellular matrix polypeptides produced uniquely by microbial cells, plant cells or mammalian cells and the like.

According to the method of the present invention, a further step of contacting said material with a second determinant comprising a biological entity is also included. Biological entities include cells or viruses, or parts thereof, wherein said cells or parts thereof are preferably mammalian cells, such as human and animal cells, plant cells, eukaryotic microbial cells, such as yeasts and fungi, and bacteria. From prokaryotic microbial cells.

If a virus or a part thereof, said virus is preferably a mammalian virus, including human and animal viruses, a plant virus, a eukaryotic microbial virus such as a yeast virus or a fungal virus, and a prokaryotic microbial virus such as a bacteriophage. Selected from microbial viruses containing Accordingly, a biological entity as defined herein preferably comprises a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion and a lipid portion and any combination thereof and the like. Such biological entities also include polyclonal antibodies, monoclonal antibodies, immunodeterminants, antigenic determinants, receptors, receptor binding proteins, interleukins, cytokines, differentiation factors, growth factors or antagonists to receptors.

The method for producing the material of the present invention is described in one preferred embodiment in US Pat.
551 (Guire). Therefore, the surface layer of the new biomaterial is
According to one preferred embodiment, it is produced in a two-step process, for example using a macromolecule amphiphile with latent (photo) reactivity. Thus, in the first step, the amphiphilic macromolecules are adsorbed to a suitable polymer substrate. A potentially reactive head group causes the amphiphile to make reactive contact with the substrate surface. The hydrophilic portion of the amphiphilic macromolecule exhibits a pronounced excluded volume resulting in a regular "self-assembled" adsorption amphiphilic macromolecule profile.

As described above, the density and pattern of the layer may depend on, for example, the amphipathic nature of the macromolecule, such as chain length and / or degree of branching, and the solution conditions (eg, concentration, solvent, salt, Temperature). Ultimately, the nature of the interface can be adjusted by changing the molecular properties of both the macromolecular substrate and the macromolecule. Similar or at least substantially similar monolayer structures can be created on very different substrates, for example by adjusting the properties of the macromolecules or the solution conditions. Amphiphilic adsorption can be easily observed by known surface physicochemical methods, such as ellipsometry or contact angle (CA) measurements.

In the second step, the excess of the macromolecule is removed and the potentially reactive head group is activated. The activation creates a covalent bond between the macromolecule and the surface of the polymer substrate. Activation is preferably performed using electromagnetic irradiation in the UV or Vis light range.

In a preferred embodiment, the method of producing the material of the present invention is performed using a macromolecule comprising a hydrophilic polymer, which is preferably poly (ethylene glycol). Macromolecules preferably have a molecular weight of greater than 400 Da. The macromolecule further comprises a conjugate comprising a bondable head group, a linker group, a polymer chain and a functional end group. The head group is preferably a photoreactive allyl azide head group. In this preferred embodiment, the substrate in contact with the macromolecule is not exposed to radiation that activates the potentially reactive head group to create a covalent bond between the substrate and the macromolecule. Without being bound by theory, it is believed that the macromolecules in contact with the substrate are anchored / immobilized on the underlying substrate by hydrophobic interactions and / or entanglement of the head group / leading group with the hydrophobic substrate. Can be

In a preferred embodiment, the method of the invention is carried out on an unpretreated substrate. Substrates, such as solid surfaces, are pre-cleaned of the surface and modified to provide the desired solvophilic properties without the addition of functional groups, for example, that cause covalent bonds with potentially reactive groups. . For example, a polystyrene surface can be cleaned and exposed to hydroxyl ions in a known water vapor plasma to add hydroxyl groups to the substrate so that the surface can be easily wetted by aqueous solutions, but the hydroxyl groups can activate potentially reactive groups. Does not participate in the formation of a covalent bond with the surface in Without the pre-treatment step identified by definition, not only would the important processing economy be avoided, but also from the technical problems associated with uniform load density deposition, bond-forming reactive groups to the surface.

EXAMPLES The following examples are examples of the present invention, and illustrate the present invention in a non-limiting manner.
Example 1.α-4-azidobenzizoyl ω-methoxypoly (ethylene glycol ) (ABMPEG)  The synthesis of the photoreactive ABMPEG 5 kDa is described. Different MWs (2,5 and 10kDa)
ABMPEG was used as a modifier in all future examples, all of which
Synthesized as described.

1. Procedure 4-Azidobenzoic acid is prepared by diazotization of 4-aminobenzoic acid with sodium nitrate ^(39,40) . The carboxylic acid is converted to 4-azidobenzoyl chloride with thionyl chloride ^(39,40) . 0.23 g of dimethylaminopyridine (DMAP) (1.875 mmol
) Is mixed with 107 ml (1.250 mmol) of trimethylamine in 10 ml of a dry methylene chloride solution. Transfer the solution to a 250 ml three neck round bottom flask. Cool to 0 ° C. and add 10 ml of a solution of 0.57 g (3.125 mmol) of 4-azobenzoyl chloride in CH ₂ Cl ₂ to give a yellow dispersion. A solution of 6.25 g (1.5 mmol) of MPEG5 kDa in dry CH ₂ Cl ₂ (50 ml) was added dropwise over 1 hour under dry nitrogen, after which the temperature was raised to room temperature. The reaction was continued overnight with stirring. The solution is filtered and ABMPEG precipitates in cold diethyl ether. The product was purified by two precipitations from CH ₂ Cl ₂ / diethyl ether and vacuum dried. Yield: 4.83 g (74%).

Embodiment 2 FIG. Adsorption properties / speed ellipsometry to ellipsometry was observed ABMPEG5kDa and MPEG5kDa polysulfo down surface is very sensitive technique to measure the adsorption rates of the optically smooth surface. For better resolution, the reflective properties of the underlying silicon were developed by spin-coating a transparent polysulfone (PSf) film on a polished silicon wafer.

[0156] 1. Preparation of PSf surface Hydrophilic silicon slide : Prepared from a polished silicon wafer in which the silica surface was thermally oxidized with pure water and saturated oxygen, and then quenched and cooled under a stream of argon to form an oxide layer of about 30 nm. did. Cut the wafer into rectangular slides (10 ~
14mm x 20-30mm), thoroughly washed with surfactant, freshly mixed sulfuric acid (96%
): Etching with a 3: 1 solution of hydrogen peroxide (30%) for 15 minutes, then complete cleaning
Fix for 2 hours and wash again with / in ultrapure water. Slides are dried at 120 ° C. for 2 hours dust-free. This procedure results in a silanol group surface density of less than 10 degrees for the contact angle.

Hydrophobic Silicon Slide : To obtain a hydrophobic surface, a previously prepared hydrophilic silicon slide is silanized in hexamethylsilazane (HMDS) saturated air at about 110 ° C. Excess HMDS is washed away with ultra pure H ₂ O. Slides are dried at room temperature and dust free. PSf-spin-coated hydrophobic silicone slide : The hydrophobic silicone slide prepared above is spin-coated with a 3% (w / w) solution of PSf in 1,2-dichlorobenzene. After the slide is completely wet with the polymer solution, 500 rpm to obtain a smooth polymer film
For 10 minutes and then at 5,000 rpm for 50 seconds. Dry the coated slides at 60 ° C. under vacuum for at least 4 hours.

[0158] 2. Polarization measurement Adsorption of ABMPEG 5kDa and monomethoxy PEG MPEG 5kDa aqueous solution onto PSf spin-coated HMDA-treated silicon slides is performed by automatic Rudolp with quartz cuvette with thermostat.
Measure in situ using an hThin Film ellipsometer, Model 43603-200E. The spin-coated slide is left to stabilize in 4.5 ml of water for at least 15 minutes or until the polarimeter and analyzer signals are steady. 0.5ml of concentrated aqueous solution of ABMPEG5kDa / MPEG5kDa
To obtain a 5 ml solution of the defined concentration.

Stir for 30 seconds with a magnetic stirrer to homogenize when adding the ABMPEG 5 kDa / MPEG 5 kDa concentrate. Polarimeter and analyzer data were collected until apparent equilibrium was achieved. From the data obtained, the thickness, refractive index and / or amount of the adsorption layer can be calculated ⁽⁴¹⁾ . When the absorption data is calculated, an approximate value is obtained for the partial specific volume and the ratio between molar weight and molar refractive index using the same value for both species for each of ABMPEG5kDa / MPEG5kDa. Since there is only an approximate value of the partial specific volume and the molar refractive index of ABMPEG5kDa / MPEG5kDa at hand, the result of the calculated adsorption amount is expressed in an arbitrary unit.

[0160] 3. Results FIG. 10 shows the adsorption velocities observed by ellipsometry for each of ABMPEG 5 kDa / MPEG 5 kDa. ABMPEG5kDa showed improved adsorption (factor = 3.5) and extended equilibration time (greater than 2 hours) when compared to MPEG5kDa. The significant difference in the adsorption properties of the two materials suggests a strong affinity between the hydrophobic (aromatic) of ABMPEG 5 kDa and the hydrophobic PSf surface.

The affinity leads the layer in a unidirectional orientation, the head group remains in close contact with the underlying substrate, and has a good positioning such that efficient grafting occurs on photoactivation. Furthermore, the amount of adsorption by flushing with water (20 ml / min) was unaffected, ie no desorption occurred in either ABMPEG 5 kDa or MPEG 5 kDa. Similar behavior is seen with other ABMPEG
Also expected for MW, for example 2 or 10 kDa. Ultimately, this example illustrates how the photoreactive head group of ABMPEG enhances the suction interaction with the hydrophobic interface, leading to increased adsorption compared to unconjugated MPEG.

Embodiment 3 FIG. ABMPEG coating of PSf spin-coated on heterogeneous glass coverslips (cover glass) by controlling hydrophilicity of polymer surface and photografting ABMPEG
Modification at 2, 5 and 10 kDa. The desired degree of hydrophilicity and thus the surface density of different ABMPEGs on PSf was achieved by adjusting the bulk ABMPEG concentration in the first adsorption step. Contact angle (CA) was used to observe the changes obtained. Different ABMP
A mixture of EG was applied to achieve intermediate surface properties. The effect of photoreactive grafting is ABMP
EG10 kDa was evaluated by removing the non-grafted ABMPEG moiety.

1. Preparation of polymer surface Glass coverslips were washed with a surfactant and ultrapure water, and then mixed with a newly mixed sulfuric acid (96%): hydrogen peroxide (30%) 3: 1 (v: v) solution. 15 at about 40 ± 5 ℃
Etch for minutes. Coverslips are thoroughly washed, fixed for 2 hours and washed again / in ultrapure water. The slip is dust free and dried at 120 ° C. for 2 hours. Wash the coverslips with n-octadecyldimethylchlorosilane (ODDMS
) Is immersed in a 2% n-hexane solution for 1 hour at room temperature to graft ODDMS.
The coverslips are washed twice with n-hexane and three times with ethanol and air dried at room temperature. The ODDMS treated cover slip is spin coated with a 3% (w: w) solution of PSf in 1,2-dichlorobenzene. Coverslips are thoroughly wetted with the polymer solution and spun at 500 rpm for 10 seconds followed by 5,000 rpm for 50 seconds to obtain a smooth surface. The coated coverslips are dried at 60 ° C. under vacuum for at least 4 hours.

[0164] 2. ABMPEG Grafting on Polymer Surfaces ABMPEG grafting involves the following two successive steps as illustrated in FIG. In the first adsorption step, different concentrations of ABMPEG aqueous solution are placed on PSf-coated coverslips, covered and left in the dark for at least 12 hours, but up to 18 hours.
The coverslips are then gently washed in ultrapure water, covered with water and immediately exposed to UV light for 1 minute. For UV irradiation, a 50W high-pressure mercury lamp with a condenser (
ORIEF). The UV-rich light was cut off at 320 nm through a high transmission glass filter to 30 mW / cm ² . Certain required control surfaces were not exposed to UV irradiation. To remove ABMPEG that is not covalently bound, a constant required sample surface is overnight, water:
After exposure to a 1: 1 (v / v) mixture of isopropanol (H ₂ O / IP), the mixture was thoroughly washed with the same mixture and ultrapure water.

[0165] 3. Contact Angle (CA) Measurements The As in modified and unmodified PS coated coverslips is measured using the captive bubble method, where air bubbles are submerged with a stainless steel needle syringe. Inject into the inverted sample surface. The diameter of the contact area between the PSf film and the bubble is always greater than 3 mm. While the needle is in the bubble, advance or sweep angle measurements can be made with a goniometer equipped with a tilt stage by gradually bleeding / adding air to the mooring bubble. In at least three places, at least 10 measurements of different bubbles are averaged to obtain one data.

[0166] 4. Results FIG. 11 shows the forward and reverse As for PSf spin coating modified with different concentrations of ABMPEG 10 kDa. The surface was exposed to UV radiation, but was not washed with (H ₂ O / IP). On the premise that the adsorbed ABMPEG layer is stable in the aqueous environment at the relevant time scale, ie no desorption occurs (see the results of Example 2), ABMPEG 10 kDa adsorption is observed and its chemical grafting is observed. Note that it is not. ABMPEG10kDa
When the bulk concentration is increased, a decrease in the forward and backward As is observed, but the CA hysteresis increases in the applicable concentration range. The results show that ABMPEG 10 kDa adsorption is very easy to control and reproducible. Desirable degree of hydrophilicity and thus ABMPEG10kD
The surface density of a can be adjusted by the bulk ABMPEG 10 kDa concentration during adsorption.

FIG. 12 shows ABMPEG of three different chain lengths, ie three different molecular weights (MWs): 2
14 shows the surface As modified by applying 5 and 10 Da (see FIG. 13). Again, the surface
Exposure to UV irradiation was not followed by H ₂ O / IP washing. Underlying PSf
The same tendency was observed for all different chain lengths for the degree of hydrophilicity and the controllability achieved in the above, but differences were found for the CA hysteresis. The CA-hysteresis value is generally lower for short chain lengths and the lowest molecular weight AM within the range applied.
BPEG has a particularly clear maximum. Thus, it appears that the longest chain lengths exhibit high CA-hysteresis, causing more chemical and / or morphological heterogeneity.

FIG. 14 shows the regression As and CA-hysteresis of PSf surfaces modified with different mixtures for ABMPEG of two different chain lengths (ABMPEG 2 kDa and ABMPEG 10 kDa). Again, the surface was exposed to UV radiation, but was not subsequently washed with H ₂ O / IP. The surface shows a gradual change in its properties. This result means that a mixture of different ABMPEG and / or ABMPEG derivatives can be applied in achieving / designing intermediate surface properties.

FIG. 15 shows the regression As of PSf surface modified with different concentrations of ABMPEG 10 kDa. The samples were exposed to UV radiation and As was measured before and after overnight washing with H ₂ O / IP. The data characterizes the efficiency of the photografting step with the applied ABMPEG concentration. Ten
For ABMPEG at a concentration greater than g / l, the effect of photoreactive grafting was H ₂ O / IP
Rapidly decreases as indicated by the reversibility of hydrophilization by washing. This indicates that a reduction in head group directivity to the surface reduces the chance of successful grafting. This may be due to the increase in solute-solute interaction with increasing surface coverage.

Embodiment 4 FIG. Assay of Protein Adsorption on Modified PSf Membranes Commercially available standard ultrafiltration membranes were modified with different degrees of ABMPEG 5 kDa modification. Thereafter, the adsorption characteristics of the membrane were evaluated by exposing the membrane to a buffer solution of bovine serum albumin (BSA) and quantifying the amount of BSA adsorbed. 1. Membrane Modification A washed piece of circularly cut PSf ultrafiltration membrane (132.6 cm ² , GR61PP type, DOW, Denmark) is immobilized and permeated through at least 6 liters of ultrapure water at 0.4 MPa for at least 1 hour. Cleaned except. Cut the membrane into a 25 mm diameter circular membrane,
The surface was modified with AMBPEG 5 kDa as described in Example 3. After exposure to UV irradiation, the membrane was exposed to ultrasound for 5 minutes and then thoroughly washed.

[0171] 2. Protein adsorption The surface layer of the unmodified or modified membrane was treated with a BSA 1 g / l solution (0.15 M phosphate buffer, p
H = 7, room temperature), flushed with buffer and dried at 60 ° C. overnight. BSA sucking
The amount deposited is determined by its total degradation and subsequent amino acid analysis⁽⁴²⁾.  3. Results FIG. 16 shows the amount of BSA adsorbed depending on the concentration of the applied ABMPEG 5 kDa. BSA adsorption, ABM
Decreases with increasing PEG 5 kDa concentration. Maximum reduction is greatest compared to unmodified standard membrane
The applied ABMPEG 5 kDA reached 70% with 10 g / l.

[0172]Example 5 .Unmodified and ABMPEG 10 kDa modified measured by in situ ellipsometry Fibronectin (FN) adsorption on PSf  As in Example 3, the reflective properties of the polished silicon are changed to unmodified or modified spin coating PS.
It was developed to observe by FN adsorption to f film. Absorption of FN on differently modified surfaces
Interfacial interaction between FN and the photografted ABMPEG 10 kDa interface structure
Bring knowledge about use.

[0173] 1. Operating procedure Fibronectin (FN) (human plasma, lyophilized, MW440kDa; Boehringer M
annheim, Germany) at a concentration of about 0.12 g / l in phosphate buffered saline (PBS; 5.8 mM NaHPO ₄ / NaH ₂ PO ₄ , 150 mM NaCl, pH = 7.4) containing 0.02% (w / v) sodium azide. did. Instrument preparation and measurement procedures are the same as described in Examples 2.1 and 2.2, respectively.

The PSf coatings on silicon wafers modified with different concentrations of ABMPEG 10 kDa as described in Example 3.1 were placed in quartz cuvettes and incubated with 2.5 ml of PBS buffer for at least 15 min or steady state of the polarimeter and analyzer signals. Fix until it is. Add 0.5 ml of concentrated FN solution to make 3 ml with a constant concentration of 0.02 g / l. When adding the concentrated protein, stir with a magnetic stirrer for 2-3 seconds to homogenize the solution. After 30 minutes, flush the PBS buffer for 10 minutes at a flow rate of 20 ml / min using the tubing pre-installed with the cuvette. Even though plateau values are typically observed after 1-2 hours (or longer), already 30 minutes can already describe protein-substratum interactions.

In calculating the amount of protein adsorbed, the different layers, silicon carrier, silicon oxide, ODMS layer, PSf coating and tethered ABMPEG 10 kDa are treated as one optical unit with an effective reflection index. The molar refractivity of FN was 3.99 g / ml when calculated as the sum of the molar refractivity of all amino acids in FN and the listed values ⁽⁴³⁾ . 0.75 ml / g was used for the partial specific volume of FN.

In FIG. 17, all data curves follow the expected monotonic rise; with flash
No FN desorption is seen. The amount of FN adsorbed decreases as the degree of ABMPEG 10 kDa surface functionalization increases, and quantitatively correlates with the decrease in CA as shown in FIG. Both unmodified and the lowest ABMPEG 10 kDa concentration, for example, PSf modified at 0.001 g / l, give approximately 1.2 μg /
A maximum adsorption of cm ² is achieved. For ABMPEG 10 kDa at a concentration of 10 g / l, the FN adsorption amount is
It is reduced by more than 60% to 0.45 μg / cm ² . As shown in Example 4, ABMPEG
BSA adsorption on the surface of the PSfUF membrane photografted with 5 kDa resulted in very similar results by total hydrolysis of the adsorbed protein, followed by amino acid analysis: the reduction ratio depends on the degree of functionalization. It correlates well with the indicated FN results.

Embodiment 6 FIG. Fibroblast Adhesion to Unmodified and ABMPEG10 kDa Modified PSf Surfaces Whole Cell Morphology, Number of Adherent Cells, and Localized Adhesion Formation The number of adherent cells, total cell morphology, and the occurrence of localized adhesions characterize the interaction between cells and interfaces. A good indicator. Many previous studies have shown that as more cells attach, more cells spread, and more pronounced localized adhesions, each surface is better suited for fixation and growth of the cells under study. showed that.

[0178] 1. Cells Human fibroblasts (HF) were collected from a new skin biopsy and used for up to 9 passages. The cells were 10% fetal bovine serum (FBS, Sigma Chemicals Co., St. Louis,
MO, USA) containing CO ₂ in Dulbecco's Modified Eagle Medium (DMEM).
They were grown using a 5% humidity incubator. HF near confluent culture
Harvested with 0.05% trypsin / 0.6 mM EDTA (Sigma), trypsin was neutralized with FBS.

[0179] 2. Adhesion HF Number and Its Morphology HF attachment was performed on 6-well culture plates containing unmodified and ABMPEG 10 kDa modified PSf coated glass slides. The experiments were performed with and without pretreatment of the surface with FBS (Sigma) at 37 ° C. for 30 minutes. Pipette about 5 × 10 ⁵ cells in DMEM into each well,
The cells were cultured at 37 ° C. for 2 hours in a humid CO ₂ incubator. The number of adherent cells and their morphology were examined and photographed directly in the hole with an inverted phase-contrast microscope Telaval31 (Carl Zeiss, Germany). The average number of adherent cells was determined by evaluating about 30 different microscopic fields randomly selected on each surface. Cell counts were normalized for: Number of microscopic fields per unit area: Standard deviation was determined for each set of surface fields.

[0180] 3. Localized adhesion formation Localized adhesion of HF on unpretreated and serum pretreated substratum was visualized by immunofluorescence. Samples were processed as follows: adherent cells were treated with formaldehyde (
3%) and infiltrated with 0.2% Triton X-100 for 5 minutes. When detecting localized adhesion, samples were treated with a monoclonal anti-vinculin antibody (Sigma Immunoch) at 37 ° C for 30 minutes.
emicals, St. Louis, MI, USA) followed by Cy3-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research, Inc. West Growe, PA, USA).
The sample was placed on a slide with Mowiol, observed, and the inverted fluorescent microscope Axiovert100 (Carl Ze
iss, Germany).

[0181] 4. Results Immediately after plating the cells (2 hours), a distinct difference can be observed depending on the underlying substratum. FIG. 18 shows the whole cell morphology of the adherent HF. A clear dependence between the amount and spread of adherent cells (see also FIG. 19) and the ABMPEG 10 kDa concentration used can be seen in the phase contrast photographs. Modified PSf at intermediate concentrations (0.001-0.01) of ABMPEG10kDA
9 shows increased cell adhesion and spread.

Even with localized adhesion formation in the base layer of the non-pre-coat illustrated in FIG. 20, the unmodified PSf on the PSf surface modified with intermediate concentrations of ABMPEG10 kDA (0.001 g / l and 0.01 g / l)
Alternatively, it showed significantly improved cell morphology and spread compared to relatively high concentrations of ABMPEG 10 kDa (1 g / l and 10 g / l). For localized adhesion formation on the serum pre-coated substrate illustrated in FIG. 21, intermediate concentrations of ABMPEG 10 kDa (0.001 g / l (B) and 0.01 g / l)
2 shows optimal localized adhesion formation in the base layer modified by. At 0.1 g / l (D), the localization has already begun to break down and at 10 g / l disappears completely. An important observation is that
The effect of ABMPEG density on localized adhesion formation is significantly more pronounced on serum-coated ABMPEG surfaces (see Figure 20 and compare Figure 21). Therefore, a small amount of ABMPEG PS
Modification of f will significantly enhance cell-substratum interactions.

Embodiment 7 FIG. Unmodified and ABMPEG10kDa modified PSf fibroblast adhesion fibronectin matrix formation fibronectin to surfaces (FN) is the adhesion protein also essential when cells adhere / fixed to any kind of substrate. Upon contact with the appropriate substratum, ready-to-grow cells secrete FN and form an FN matrix. The amount of secreted FN and the structure of the subsequently formed matrix can be used to assess the nature of the cell-substratum interaction.

1. Formation of FN Matrix Approximately 5 × 10 ⁵ HFs in 3 ml of medium containing 10% FBS were applied to 6-well cell culture plates containing PSf-coated and photo-modified glass slides (Falcon, Becton Dickenson & Com.
pany, New Jersey, USA) for 5 days. After culturing, the cells were fixed with 3% paraaldehyde and the FN matrix deposited on different surfaces was treated with a specific anti-human FN matrix mouse monoclonal antibody (lot No. 0326, ImmunotecHSA, France) and then Cy3-conjugated. Goat anti-mouse second IgG1 antibody (Jackson Immuno Researc
h, Inc. West Growe, PA, USA). Further examination and photographing were conducted using the inverted fluorescence microscope described above.

[0185] 2. Results FIG. 22 shows the maximum FN production of HF cultured on medium ABMPEG 10 kDa density surfaces. Note that secreted FN is highly organized on these surfaces (see FIG. 23).

Embodiment 8 FIG. Fibroblasts, unmodified PSf and ABMPEG10kDa modified growth human fibroblasts unmodified and ABMPEG10kDa modified PSf table surface of hepatocytes and endothelial cells (HF), hepatocytes (C3A) and human umbilical vein endothelial cells (HUVEC) Phase contrast photographs were taken to characterize the morphology and growth of whole cells on PSf. For HF proliferation, further characterization by established semi-quantitative XTT and LDH assays for functional methods of cell proliferation was performed. The XTT assay is an assay based on the reductive cleavage of a water-soluble tetrazole salt by dehydrogenase activity of intracellular intact mitochondria, and can be quantitatively followed by color change. The LDH assay is an assay that directly monitors the activity of lactate dehydrogenase released by cells.

[0187] 1. Polymer surface As described in Examples 3.1 and 3.2, glass cover slips (15 × 15 and 18 ×
18mm ² ) and slides (26 × 76mm ² ) are cleaned, hydrophobized, PSf spin coated, ABM
PEG was modified by 10 kDa. The surface was stored in a 0.02% NaN ₃ solution, washed with distilled water before applying the cells, immersed in 70% ethanol for 10 seconds, and air-dried under sterile conditions. Cover slips (15 × 15 mm ² ) were loaded into 12-well plates and the larger coverslips into 6-well plates. Slides were demarcated by applying a Flexperm silicon mask that splits the polymer surface into 8-wells.

[0188] 2. Cell and Proliferation Considerations HFs were cultured and harvested as described in Example 6.1. For HUVEC and C3A, another medium (endothelial cell growth medium for HUVEC and MEM for C3A) was used, but the other culture and culture were performed in the same manner as HF. After centrifugation and resuspension, cell numbers were counted in Neubauer counting chamber. The following cells and densities were applied for cell growth considerations.

[0189]

[Table 1] Cells were seeded in wells and incubated at 37 ° C. and 5% CO ₂ for up to 7 days. Samples after 3, 5, and 7 days were visually inspected to demonstrate morphology / state by phase contrast photography. After 1, 3, and 7 days, XTT and LDH assays were performed.

[0190] 3. Results For all three types of cells, the best growth conditions were adjusted to a concentration range of 0.01 g / L to 0.1 g / L.
g / L of ABMPEG10 kDa modified PSf (FIG. 24 for HF, FIG. 25 for HUVEC, C
See Figure 26 for 3A). Both HF and C3A grew very well during the 7-day culture period and grew almost completely over the culture substrate at the indicated ABMPEG intermediate concentrations. HF was found to grow in multiple layers. The number of HUVECs was low due to the low initial seeding density (22,0 for HF and HUVEC).
00 cells / cm ² or more in against 5,500 cells / cm ²⁾ was lower because of the.

XTT and LDH assays performed on HF confirmed the observed trend (see FIGS. 27 and 28), ie, maximum growth on PSf surfaces modified at intermediate concentrations of ABMPEG 10 kDa. However, these assays showed significantly smaller maxima as compared to the phase contrast photographs. This is probably due to the boundary effect derived from the Flexiperm silicon well used. Significant cell attachment and proliferation was observed at the contact line between the silicon and the underlying PSf culture substrate.

Embodiment 9 FIG. Centralized adhesion formation on unmodified and ABMPEG10 kDa modified PSf of endothelial cells The occurrence of centralized adhesion is a measure of the quality of the interaction between the cell and the interface (see Example 6). 1. Cell culture The surface studied was the FHN precoated surface (see reference 13 for details). HUVECs were cultured and coated as described in Example 8. Immunofluorescence considerations were performed as described in Example 8 to visualize the convergence points of attachment between HUVEC and the underlying culture substrate. After a 2 hour incubation, cells were examined by phase contrast microscopy and then fixed with 3% paraformaldehyde in PBS for 15 minutes. Further characterization was performed as described in Example 6.3.

[0193] 2. Results FIG. 29 clearly shows the same sample dependence of concentrated adhesion formation as already seen in previous growth and adhesion studies on the extent of ABMPEG 10 kDa modification of the PSf culture substrate. HUVECs modified with an intermediate concentration of ABMPEG and applied to PSf culture substrates are the largest number of concentrated attachments and those that occurred most often.

Embodiment 10 FIG. Evaluation of the interaction of proteins adsorbed on different surfaces with their antibodies by circular dichroism.The binding of antibodies to their antigens is both, i.e. when the antigens and antibodies are present in their native or bioactive conformation. Only valid for. Antigen / antibody binding usually shows tight junctions with a high dissociation constant. As discussed above, protein adsorption often loosens the integrity of its higher-order structure and thus loses its ability to bind to each antibody [44]. Some studies have monitored these changes in conformation / antibody binding to monitor either the release of bound antigen [45,46] or the respective antibody binding to the pre-adsorbed antigen, for example with a circular dichroometer. [47, 48].

This example presents data indicating that the binding affinity of the adsorbed proteins (antigens) for each antibody increases when these antigens are adsorbed to a previously ABMPEG-modified interface. Bovine immunoglobulin (BGG) and enzyme-labeled anti-BGG (H + L) are selected as model systems. Enzyme-labeled anti-BGG (H + L) is an antibody directed toward the heavy and light chains of BGG, ie, four independent epitopes present on each BGG molecule. Further, human serum albumin (HSA) was applied as a blocking agent to cover the remaining surface adsorbed portion before exposing the anti-BGG to the preadsorbed BGG.

[0196] 1. Preparation of Surface Hydrophilic, hydrophobic and PSf spin-coated silicon slides were prepared as described in Example 2.1. PSf spin-coated slides were prepared using ABMPEG as described in Example 3.2.
The transplant was performed at a transplant density range of 10 kDa. 2. Material BGG-FITC: Fluorescent (FITC) composite Chrom Pure BGG (Lot 001-090-003, Jackson
Immuno-Research Laboratories, Inc., a-BGG (H + L) column purification), antigen, abbreviated as BGG a-BGG (H + L) -HRP: BGG (Southern Biotechnology Associates, Inc.)
Horseradish peroxidase (HRP) conjugated goat anti-bovine BGG (H + L) from goat hyperimmune antiserum pool, antibody, abbreviated as a-BGG HSA: human serum albumin (Centeon Pharma GmbH), blocking agent

[0197] 3. (I) Antigen B on unmodified and ABMPEG-modified PSf spin-coated silicon slides
Circular dichroism determination of continuous adsorption of GG, (ii) blocking agent HAS, and (iii) each BGG antibody a-BGG. The setting of the instrument and the principle of measurement operation are described in Example 2.2. Differently modified PSf spin-coated silicon slides were placed in quartz cuvettes and 2.5 ml of phosphate buffered saline (PBS; 154 mM NaCl, 10 mM Na ₂ HPO ₄ / PBS) until constant polarizer and analyzer signals were obtained.
NaH ₂ PO ₄ , pH = 7.4). Previously purified protein, BGG, a-BGG
And HAS were re-established in PBS. At the start of each experiment (0 min), 0.5 ml of BGG-
The FITC solution was added to the cuvette to obtain 3 ml of a solution having a specified concentration of 0.01 g / L. The contents of the cuvette were continuously stirred with a magnetic stirrer during the experiment.

30 minutes after BGG adsorption, 20 ml / min of PBS was added using a tube pre-loaded with cuvettes.
Flush at flow rate for 1 minute. Thereafter, the total volume in the cuvette was readjusted to 2.5 ml. After another minute (signal stabilization period), 0.5 ml of concentrated HAS solution was added (to 32 minutes).
3 ml of a 3 g / L solution having a specified concentration was obtained. Ten minutes after HAS adsorption, the cuvette was flushed with PBS and the total volume was readjusted to the above amount. At 44 minutes, 0.25 ml of the concentrated a-BGG solution was added to obtain 2.75 ml of a specified concentration of 0.015 g / L solution. 60 minutes after a-BGG adsorption (
At 104 minutes) the cuvette was flushed with PBS buffer for 2 minutes and finally waited 1 minute for signal stabilization. The experiment was terminated at 107 minutes.

Polarizer and analyzer data are collected automatically over the entire period and the corresponding Ψ and Δ values are calculated directly. The relative change in the Ψ signal (the change in the Ψ signal is proportional to the total mass adsorbed) calculated during the stabilization period in PBS is used to compare the amount of adsorption of different successive adsorbed proteins. All data presented are arithmetic means of two independent experiments.

[0200] 3. Experiments with different controls Sequential adsorption of BGG, HSA and a-BGG was performed on hydrophobic, hydrophilic silicone slides as described in the previous paragraph. On some selected ABMPEG-modified PSf spin-coated silicon slides, the first adsorption step, ie, the adsorption of antigen BGG, was not performed. However, the adsorption of HSA and a-BGG was performed as described in the previous paragraph.

[0201] 4. Results Two total adsorption experiments on unmodified PSf and ABMPEG 10 g / L modified PSf, namely BGG
, HSA and the continuous adsorption of a-BGG are shown in FIG. With unmodified PSf, the kinetics of BGG adsorption levels off after approximately 20 minutes with an almost constant value of Ψ = 0.40. As shown by the first arrow, after 30 minutes, the PBS flush begins, followed by the addition of HSA (second arrow) to act as a blocking agent to cover the BGG uncoated remaining surface area. The applied HSA concentration was 300 times higher than the applied BGG concentration, but between two PBS flushes (first and third arrows)） 0.27.
Only the unit rises, indicating that the polymer interface is already substantially saturated with BGG.

Furthermore, the rapid leveling of the Ψ signal associated with the addition of HSA, and the small size and high adhesion of the protein compared to BGG indicate a blocking effect of the remaining uncoated interface. However,
Re-addition of low concentrations of a-BGG (arrow 4) followed by a flash (between arrows 5 and 6) resulted in a significant increase in the Ψ signal (0.52 units). This increase can be attributed to the high affinity of the antigen-antibody binding and is due to the second protein layer formed on the adsorbed BGG / HSA layer. The relatively weak kinetics of a-BGG binding is a further indicator of the different binding mechanism of this second layer, ie, antigen-antibody binding to adsorptive binding.

At 10 g / L of PEG-modified PSf-ABMPEG, the progress of BGG adsorption is rather slow, to a much smaller extent (0.035 units). ABMPEG, already present and covalently fixed, approaches B
The steric hindrance of the ABMPEG moiety to the GG molecule reduces the available surface and also reduces the rate of adsorption. However, as described above, HSA, which is much smaller than BGG and is a more adhesive protein, has less adsorption inhibition between the existing ABMPEG portions,
Thus, the adsorption is about the same as on PSf covered with BGG only (0.33 units) (see above).

The continuous binding of a-BGG is much smaller (0.15 units) than the continuous binding with unmodified PSf (0.52 units). However, the ratio between the bound a-BGG and the BGG, but not the total amount of a-BGG bound to the surface, specifies the binding affinity of the already adsorbed BGG. However, this ratio increased from 1.2 unmodified PSf to PEG-modified PSf-AB in this experimental group.
MPEG increased to 4.3 g of 10 g / L, indicating higher binding affinity, and thus higher conformational integrity or BGG bioactivity when adsorbed to PEG-modified PSf.

FIG. 31 (ac) shows the arithmetic mean of the relative increase in the Ψ signal for the continuous adsorption of BGG, HSA and a-BGG for all experiments performed with unmodified PSf (ref) and ABMPEG modified PSf. . Error bars indicate standard deviation of duplicate experimental groups. As expected, BGG adsorption decreases by approximately 95% from 0.5-0.03 Ψ-units as ABMPEG engraftment density increases (see FIG. 31 (a)). Continuous adsorption of a relatively high concentration of HSA gives the maximum value with ABMPEG-modified PSf when the concentration is 1 g / L (FIG. 31 (b)). a-BGG adsorption decreases by approximately 75% from 0.62 to 0.15 ° units as ABMPEG implant density increases (FIG. 31 (c)).
). Therefore, the ratio of the amount of a-BGG bound to the amount of BGG already adsorbed is
(Ref) from 1.25 to 5.0 of PSf-ABMPEG-10 g / L (the latter value is the arithmetic mean of two independent experimental groups).

In a control experiment with hydrophobic wafers, unmodified PSf (BGG = 0.39 ± 0.08, HSA = 0.17)
± 0.02, a-BGG = 0.40 ± 0.05).
Is 1.03 ± 0.08, slightly lower than the PSf result. Control experiments performed on hydrophilic wafers did not yield definitive results because the amount of adsorbed protein was too low.

To verify the effect of the blocking agent, HSA, ABMPEG modified PSf slides (0.01 and
1.0 g / L ABMPEG) under the same conditions as in the previous adsorption experiment, ie 3 g / L
L was directly exposed to HSA for 10 minutes in a PBS buffered HSA solution. Continuous exposure to the a-BGG solution (the procedure described above, ie, a-BGG: 0.015 g / L, adsorption time: 60 minutes) resulted in a slight increase (0.1 units) in the 単 位 signal for both surfaces. However, when comparing this increase with the increase obtained in the presence of the already adsorbed BGG (see FIG. 31 (c)), a much larger increase in a-BGG adsorption was observed, averaging an increase of 0.38 units, ie A value four times higher was obtained.

Several conclusions can be drawn from these data. (I) The adsorption of BGG to ABMPEG-modified PSf decreases as the degree of ABMPEG surface modification increases, as observed for FN and BSA in Example 5 (see FIG. 31 (a)). This is AB
It confirms that increasing the density of MPEG transplantation reduces protein adsorption on these surfaces, independent of the adsorbed protein (FN, BSA or BGG). (Ii) Under the following assumptions: (a) HSA effectively blocks the interface available for further adsorption, (b) a-BGG is also implanted in adsorbed HSA (as shown) Does not bind to ABMPEG to a high degree (estimable, see references 14 and 33), adsorbed BG
The increase in the ratio of adsorbed a-BGG to G can be described as higher order structural integrity or increased bioactivity of adsorbed BGG.

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olymer properties), J Biomed Mater Res 24, 1263-1287 (1990) [47] Nygren H, "Real-time recording of antigen-antibody reactions on surfaces: Interpretation of data and statistical models (Real-time recording of antigen- antibody reactions at s
urfaces: interpretation of data and a statistical model), Colloids Su
rf, B4, 243-250 (1995) [48] Nygren H, Kaartinen M, Stenberg M. "Determination by ellipsometry of the affinity of monoc
lonal antibodies) ", J Immunol Methods 92, 219-225 (1986)

[Brief description of the drawings]

FIG. 1 shows that the conformation of an absorbed / immobilized biologically active moiety (eg, a polypeptide) changes (over time), and then the lower substrate and the absorbed / immobilized polypeptide FIG. 3 is a schematic diagram of inactivation due to attractive (eg, hydrophobic) interactions between them.

FIG. 2 is a schematic diagram showing how to measure contact angles in three boundary phases, namely, a base layer (solid), water (liquid), and water vapor (gas), in which water vapor bubbles are immersed in water. Trapped by the upper horizontal substrate.

FIG. 3 shows that an immobilized hydrophobic polymer adjacent to a portion (eg, a polypeptide) that is absorbed / immobilized in the gap is placed in an active conformation by applying lateral pressure to the portion. It is the schematic which stabilizes.

FIG. 4 is a schematic diagram of a two-stage polymer surface functionalization procedure.

FIG. 5 is a schematic diagram in which a hydrophilic polymer adjacent to a portion absorbed / immobilized in a gap is immobilized on a lower base layer by a chemical bonding method.

FIG. 6 is a schematic diagram in which a hydrophilic polymer adjacent to a portion absorbed / immobilized in a gap is immobilized on a lower base layer by a chemical bonding method (charge).

FIG. 7 is a schematic diagram in which a hydrophilic polymer adjacent to a portion absorbed / immobilized in a gap is immobilized on a lower base layer by an absorbing means.

FIG. 8 is a schematic diagram in which a hydrophilic polymer adjacent to a portion absorbed / immobilized in a gap is immobilized on a lower polymer base layer by means of mutual entanglement.

FIG. 9 shows that the hydrophilic polymer adjacent to the portion absorbed / immobilized in the gap is the lower SA
It is the schematic which is fixed to M.

FIG. 10. ABMPEG 5 kDa and MPEG from aqueous solution (10 g / l) onto PSf, spin-coated on polished silica wafers as determined by ellipsometry.
FIG. 4 is an absorption rate diagram of 5 kDA.

FIG. 11 is a diagram of CA back and forth on PSf spin-coated on glass cover pieces and modified with various ABMPEG 10 kDa bulk concentrations.

FIG. 12 is an illustration of CA backing on PSf spin-coated on glass cover pieces and modified with different concentrations of ABMPEG 10 kDa, ABMPEG 5 kDa and ABMPEG 2 kDa and their hysteresis.

FIG. 13 is a table of CA hysteresis and backward CA associated with FIG. 12 (nd:
No data available).

FIG. 14 is an illustration of CA back and forth on PSf modified with a mixture of ABMPEG 2 kDa and ABMPEG 10 kDa spin-coated on glass cover pieces to yield a total ABMPEG concentration of 10 g / l.

FIG. 15 is a diagram of CA backing over PSf spin-coated on glass cover pieces and modified with various concentrations of ABMPEG10 kDa, modified, and subsequently rinsed with isopanol / water = 1/1. Shows the CA after leaving.

FIG. 16 shows that the membrane was placed in a 1 g / l BSA solution (0.15 M phosphate buffer, pH = 7, room temperature).
Time static exposure followed by gentle rinsing with buffer followed by unmodified and ABMPEG 5 kD
FIG. 4 is a diagram of BSA absorption on a modified PSfUF membrane.

FIG. 17 shows unmodified and ABMPEG10 kDa-modified PSf spin-coated on polished silicon wafers as monitored by in-situ ellipsometry.
FIG. 3 is an absorption rate diagram of FN with respect to.

FIG. 18 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass cover pieces.
FIG. 2 is a morphology diagram of whole cells of HF adhering to modified PSf. Effect of ABMPEG 10 kDa density. HF
Represents various concentrations on unmodified PSf (A) or as follows, (B) 0.001 g / l, (
C) 0.01 g / l, (D) 0.1 g / l, (E) 1 g / l, (F) 10 g / l ABMPEG1
A membrane was formed for 2 hours on PSf implanted at 0 kDa. At the end of the culture, the samples were examined and photographed under low magnification (20x) phase contrast.

FIG. 19 shows unmodified PSF and ABMPEG10 kDa spin-coated on glass cover pieces.
FIG. 4 is a diagram of the number of attached HF per microscope field on modified PSf. Error bars represent the standard deviation of the data obtained.

FIG. 20 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass cover pieces.
FIG. 4 is a focal formation diagram of HF foci adhering to modified PSf. Effect of ABMPEG 10 kDa density. HF
Are unmodified PSf (A) or various concentrations as follows, (B) 0.001 g / l, (C
ABMPEG10k of 0.01 g / l, (D) 0.1 g / l, (E) 1 g / l, (F) 10 g / l
A membrane was formed on the PSf implanted with Da for 2 hours. At the end of the culture, cells were fixed, allowed to penetrate, and stained for vinculin by immunoluminescence. Samples were visualized and photographed at high magnification (100 ×).

FIG. 21 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass cover pieces.
FIG. 4 is a focal formation diagram of HF foci adhering to modified PSf. Effect of serum pre-coating. HF was obtained from serum-coated unmodified PSf (A) or various concentrations such as:
) 0.001 g / l, (C) 0.01 g / l, (D) 0.1 g / l, (E) 1 g / l, (F)
Membranes were formed for 2 hours on serum-coated PSf implanted with 10 g / l ABMPEG 10 kDa.
At the end of the culture, the samples were fixed, infiltrated and stained for vinculin. Samples were visualized and photographed at high magnification (100 ×).

FIG. 22 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass cover pieces.
FIG. 2 is a diagram showing the formation of an FN matrix by HF cultured on a modified PSf. Effect of PEG density.
HF was obtained from (A) unmodified PSf or various concentrations as follows, (B) 0.001 g / l,
C) 0.01 g / l, (D) 0.1 g / l, (E) 1 g / l, (F) 10 g / l ABMPEG1
Cultured on modified PSf implanted at 0 kDa for 5 days. At the end of the culture, HF was fixed and stained for FN by immunoluminescence. Samples were visualized and photographed at low magnification (25x).

FIG. 23. FN by HF cultured on unmodified PSf and ABMPEG10 kDa modified PSf.
It is a matrix formation figure. Effect of ABMPEG 10 kDa density. HF was added to (A) unmodified PSf or various concentrations as follows, (B) 0.001 g / l, (C) 0.01 g / l, (D) 0.1
g / l, (E) 1 g / l, (F) 10 g / l ABMPEG 10 kDa on modified PSf
The cells were cultured in DMEM containing 10% FBS for 5 days. At the end of the culture, HF was fixed and stained for FN by immunoluminescence. Samples were visualized and photographed at high magnification (100 ×).

FIG. 24 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass slides.
FIG. 4 is a diagram of HF growth on modified PSf. Phase contrast pictures were taken on days 1, 3, and 7.

FIG. 25 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass slides.
FIG. 4 is a diagram of HUVEC growth on modified PSf. Phase contrast photographs were taken on days 3, 5, and 7.

FIG. 26 shows unmodified PSf and ABMPEG10 kDa spin-coated on glass slides.
FIG. 4 is a diagram of C3A growth on modified PSf. Phase contrast photographs were taken on days 3, 5, and 7.

FIG. 27. Day 1, 3 and 7 cultured on unmodified PSf and ABMPEG10 kDa-modified PSf spin-coated on glass slides and sectioned by silicon mask into 8 wells. FIG. 7 is a diagram of a later XTT assay for HF. Error bars represent the standard deviation of the data.

FIG. 28. Day 1, 3 and 7 cultured on unmodified PSf and various ABMPEG10 kDa-modified PSf spin-coated on glass slides and partitioned into eight wells with a silicon mask FIG. 4 is a diagram of an LDH assay for HF immediately after. Error bars represent the standard deviation of the data.

FIG. 29 is a focal adhesion diagram of HUVECs adhering to unmodified and ABMPEG10 kDa modified PSf spin-coated on glass cover pieces. HUVECs are FN-coated unmodified PSf (A) or various concentrations as follows, (B) 0.001 g / l, (C) 0.
A membrane was formed for 2 hours on FN-coated PSf transplanted with ABMPEG10kDa of 01g / l, (D) 0.1g / l, (E) 1g / l, (F) 10g / l. At the end of the culture, the samples were fixed, infiltrated and stained for vinculin. Visualize the sample at high magnification (100 ×
).

FIG. 30 shows i) BGG as antigen, ii) HSA as blocking agent and iii) unmodified PSf of a-BGG as the respective antibody to BGG, and ABMPEG at a concentration of 10 g / l.
FIG. 4 is a diagram of ellipsometric data of continuous absorption for PSf modified with 10 kDa. PS
f was previously spin-coated on polished silicon slides. Arrows indicate rinsing with buffer described in the text or addition of concentrate.

FIG. 31 shows panels a) BGG as a antigen, b) HSA and c as blocking agents.
A) Unmodified PSf of a-BGG as the respective antibody against BGG and various concentrations of ABM
Fig. 3 shows ellipsometric data of continuous absorption for PSf modified with PEG10kDa. Error bars represent each standard deviation. PSf was previously spin-coated on polished silicon slides. FIG. 31d shows the ratio between the amount of a-BGG absorbed in the third stage (c) and the amount of BGG bound in the first stage (a).

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──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID , IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Ulbricht, Matthias Germany, Day 10407 Berlin, Landsberger Alley 89 (72) Inventor Jankova, Katoya, Bulgaria, 8001 Burgas, Todov Kabreshkov Street 12B, Complete. Lazul 11 (72) Inventor Altankov, Jorge Bulgaria, 1113 Sofia, Yoriot Curie 75, Bier. 313 F term (reference) 4C081 AB31 BA17 CA02 CA18 DA02 DB08

Claims (146)

[Claims] 1. A material having a substrate, wherein the substrate is capable of contacting a macromolecule, the material further comprises at least one macromolecule, the material has a first contact angle a, and the substrate is A second contact angle b ₀ when not in contact with the macromolecule, and another second contact angle b _sat when the substrate is saturated with the macromolecule as defined herein; The relationship between the contact angles is the ratio R = (b ₀ −a) / (b ₀ −b _sat )
Wherein the value of R is in a range of 0 to less than 0.6 including 0, wherein the material includes the substrate. 2. The material comprises a substrate, wherein the substrate is capable of contacting a macromolecule, wherein the material further comprises at least one macromolecule, wherein the material has a first contact angle a, The material of claim 1, wherein the material has a second contact angle b ₀ when the substrate is not in contact with the macromolecule, and the contact angle a is substantially the same as the contact angle b ₀ . 3. A material having a substrate having a first contact angle and having a second contact angle, wherein the substrate is in contact with a macromolecule, and (i) when the macromolecule is absent. The difference between the second contact angle and the first contact angle; (ii) the second contact angle in the absence of macromolecules and the contact of the substrate when the substrate is saturated with the macromolecules defined herein. A first contact angle, wherein the relationship between the first and second contact angles defined by a ratio between the first and second contact angles is greater than -0.6 and less than 0.6; And a material including a substrate having a second contact angle. 4. A material comprising a substrate having a first contact angle and having a second contact angle, wherein said substrate is capable of forming a self-assembled monolayer containing macromolecules. (I) the difference between the third contact angle and the first contact angle of the single layer when no macromolecule is present; and (ii) the difference between the single contact layer when no macromolecule is present. A difference between the third contact angle and the contact angle of the self-assembled monolayer when the monolayer is saturated with the macromolecules defined herein; A material having a first contact angle and comprising a substrate having a second contact angle, wherein the relationship is greater than -0.6 and less than 0.6. 5. The material of claim 2, wherein said soluble material is selected from the group consisting of molecules capable of forming a self-assembled monolayer. 6. The material according to claim 1, wherein the substrate has been pretreated or modified. 7. The material according to claim 4, wherein the pretreated or modified substrate is in contact with and / or effectively linked to a charged group or a hydrophilic compound. 8. The material according to claim 1, wherein said first contact angle is an advancing contact angle. 9. The material of claim 6, wherein said first contact angle ranges from 50 degrees to 140 degrees. 10. The material according to claim 6, wherein said first contact angle ranges from 60 degrees to 125 degrees. 11. The material of claim 6, wherein said first contact angle ranges from 70 degrees to 120 degrees. 12. The material according to claim 6, wherein said first contact angle ranges from 75 degrees to 110 degrees. 13. The material according to claim 6, wherein said first contact angle ranges from 80 degrees to 100 degrees. 14. The material of claim 6, wherein said ratio is less than 0.50. 15. The material of claim 6, wherein said ratio is less than 0.40. 16. The material according to claim 6, wherein said ratio is less than 0.30. 17. The material of claim 6, wherein said ratio is less than 0.25. 18. The material of claim 6, wherein said ratio is less than 0.20. 19. The material of claim 6, wherein said ratio is less than 0.15. 20. The material of claim 6, wherein said ratio is less than 0.10. 21. The material of claim 6, wherein said ratio is less than 0.05. 22. The material according to claim 1, wherein the first contact angle is a receding contact angle and the ratio is less than 0.40. 23. The material of claim 20, wherein said first contact angle ranges from 30 degrees to 120. 24. The material of claim 20, wherein said first contact angle ranges from 40 degrees to 110 degrees. 25. The material according to claim 20, wherein said first contact angle ranges from 50 degrees to 100 degrees. 26. The material according to claim 20, wherein said first contact angle ranges from 60 degrees to 90 degrees. 27. The material of claim 20, wherein said first contact angle ranges from 70 degrees to 80 degrees. 28. The material according to claim 20, wherein said ratio is less than 0.35. 29. The material of claim 20, wherein said ratio is less than 0.30. 30. The material of claim 20, wherein said ratio is less than 0.25. 31. The material of claim 20, wherein said ratio is less than 0.20. 32. The material according to claim 20, wherein said ratio is less than 0.15. 33. The material of claim 20, wherein said ratio is less than 0.10. 34. The material of claim 20, wherein said ratio is less than 0.05. 35. When the material is contacted with a first determinant comprising a compound selected from the group comprising a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion, a lipid portion and any complex thereof. 35. The material according to any one of claims 1 to 34, wherein the material is capable of maintaining the compound in a bioactive form. 36. The method according to claim 33, wherein the compound is a polypeptide or a part thereof.
The material described in the above. 37. The material further comprising the first determinant comprising the compound, wherein the first determinant is maintained in a bioactive form when contacted with the substrate and / or the macromolecule. 35. The material according to claim 33 or claim 34, characterized by the fact that: 38. The material of claim 35, wherein said bioactive form is essentially a bioactive conformation. 39. The material according to any of claims 33 to 36, wherein the bioactive form or higher order structure is maintained and / or improved and / or stabilized by means of the cooperative action of the substrate and the macromolecule. . 40. The bioactive form or higher order structure is maintained and / or improved and / or stabilized when contacted with the substrate and the macromolecule.
Material according to to 37. 41. A material according to any of the preceding claims, wherein said material is biocompatible. 42. The weight gain per unit area resulting from a portion of a macromolecule consisting essentially of PEG or polyethylene oxide (PEO) per square nanometer (nm ² )
A material according to any of the preceding claims, wherein the material is 2.0 x ^10-22 grams (g). 43. The material of claim 40, wherein said difference is 1.0 × 10 ^-22 grams per square nanometer (nm ² ). 44. The material of claim 40, wherein said difference is 0.8 × 10 ⁻²² grams (g) per square nanometer (nm ² ). 45. The material of claim 40, wherein said difference is 0.5 × 10 ⁻²² grams (g) per square nanometer (nm ² ). 46. The material of claim 40, wherein said difference is 0.3 × 10 ⁻²² grams (g) per square nanometer (nm ² ). 47. The material according to any of the preceding claims, wherein said substrate is in contact with a plurality of soluble compounds capable of forming a self-assembled macromolecular layer. 48. The composition according to claim 48, wherein said soluble compound preferably contains from 8 to 24 carbon atoms.
46. The material according to claim 45, which is an alkane chain. 49. The material according to any of the preceding claims, wherein each macromolecule is associated with an excluded volume. 50. The material according to any of the preceding claims, wherein said substrate comprises a hydrophobic polymer. 51. The material of claim 48, wherein said substrate is at least substantially flexible. 52. The material according to claim 48, wherein said substrate is a film. 53. The material of claim 45, wherein said substrate is essentially rigid or at least substantially inflexible. 54. The method of claim 51, wherein said substrate comprises a crystalline structure, such as gold, silicon oxide, a nanometer-scale smooth, similar crystalline structure and / or structure capable of supporting a self-assembled monolayer. material. 55. The material of any of claims 1 -54, wherein said macromolecule comprises a hydrophilic polymer. 56. The material of claim 53, wherein said macromolecule comprises an amphiphilic polymer. 57. A material according to any of the preceding claims, wherein the macromolecule has a molecular weight of more than 400 Da. 58. The material of claim 55, wherein said macromolecule has a molecular weight greater than 1,000 Da. 59. The material of claim 55, wherein said macromolecule has a molecular weight greater than 5,000 Da. 60. The material of claim 55, wherein said macromolecule has a molecular weight greater than 10,000 Da. 61. The material of claim 55, wherein said macromolecule has a molecular weight of greater than 50,000 Da. 62. The macromolecule has a molecular weight of greater than 100,000 Da.
The material described in the above. 63. The material according to any of the preceding claims, wherein said macromolecule is a conjugate comprising a head group, a guide group, a linker group, a polymer chain or a main body and a functional end group. 64. The material of claim 61, wherein said head group is capable of forming a chemical bond. 65. The material of claim 61, wherein said head group is capable of adsorbing to a substrate. 66. The material of claim 61, wherein said head group is capable of forming an ionic bond. 67. The material of claim 61, wherein said head group can be included in or with a substrate. 68. The head group can form a self-assembled monolayer.
61. The material according to 61. 69. The material according to claim 61, wherein said guide group is a bifunctional group containing an aliphatic, linear or slightly branched group. 70. The material of claim 61, wherein said linker group is enzymatically or chemically hydrolysable. 71. The material of claim 61, wherein said linker group is hydrolytically labile. 72. The material of claim 61, wherein said linker group is essentially stable to cleavage in a practical environment. 73. The material of claim 61, wherein said polymer chains or entities are hydrophilic in an aqueous environment, preferably do not unwind, and exhibit an excluded volume. 74. The material of claim 64, wherein said functional end group is capable of permanently or reversibly linking another biomolecule, synthetic molecule or material. 75. The method of any of claims 33 to 72, wherein said first determinant comprises a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion, a lipid portion, and a bioactive compound including any complex thereof. The material described in Crab. 76. The material of claim 73, wherein said bioactive compound comprises a polypeptide. 77. The material according to claim 73, wherein said bioactive compound is selected from the group consisting of a membrane-associated and / or extracellular matrix polypeptide native to microbial, plant, or mammalian cells. 78. The bioactive compound may be a polypeptide, antibody, polyclonal antibody, monoclonal antibody, immunogenic determinant, antigenic determinant, receptor, receptor binding protein, interleukin, cytokine, cell differentiation factor, cell growth. 74. The material of claim 73, selected from the group consisting of a factor and a receptor antagonist. 79. The material of claim 73, wherein said bioactive compound is a synthetic peptide or a portion thereof capable of contacting said substrate and / or said macromolecule. 80. The material of claim 73, wherein said bioactive compound is a synthetic peptide or a portion thereof capable of contacting said substrate and said macromolecule. 81. The material according to claim 73, wherein said bioactive compound is an attachment polypeptide, preferably fibronectin or vitronectin. 82. A biological cell wherein said bioactive compound comprises said material and a polypeptide or a part thereof, or an organism comprising a virus or a part thereof, a nucleic acid part, a hydrocarbon part, a lipid part and any complex thereof. 80. The material according to any one of claims 33 to 79, which provides improved contact with a target entity. 83. A material according to any of the preceding claims, wherein said material further comprises a second determinant. 84. A biological entity such as a biological cell or virus, or a part thereof, a nucleic acid part, a hydrocarbon part, a lipid part and a complex thereof, wherein the second determinant contains a polypeptide or a part thereof. 81. The material of claim 81, comprising: 85. The biological entity is a polypeptide, antibody, polyclonal antibody, monoclonal antibody, immunogenic determinant, antigenic determinant, receptor, receptor binding protein, interleukin, cytokine, cell differentiation factor, cell growth. 83. The material of claim 81, wherein the material is selected from the group consisting of a factor and a receptor antagonist. 86. The biological cell or a part thereof is selected from the group consisting of mammalian cells including human cells and animal cells, plant cells, microbial cells including eukaryotic microbial cells including yeast and fungi, and prokaryotic cells including bacteria. 83. The material of claim 82 wherein said material is: 87. The material according to claim 84, wherein said biological cells are mammalian cells. 88. The virus or part thereof consists of mammalian viruses including human and animal viruses, plant viruses, microbial viruses including eukaryotic microorganisms including yeast viruses and fungal viruses, and prokaryotic microorganisms including bacteriophages. 83. The material of claim 82 selected from the group. 89. The material of claim 86, wherein said virus is a mammalian virus. 90. The material according to any of the preceding claims, wherein said substrate is porous, preferably a membrane. 91. The material of claim 88, wherein the flux of water through the material is substantially unchanged compared to the flux of water through the porous substrate. 92. The material according to claim 1, wherein the substrate is non-porous and / or substantially water-impermeable. 93. A material according to any of the preceding claims for use in a method of controlling cell growth and / or proliferation and / or differentiation in vitro. 94. A material according to any of the preceding claims for use in a method of separating and / or isolating a biomaterial in vitro. 95. A material according to any of the preceding claims for use in a method of producing an in vitro biohybrid organ. 96. A material as claimed in any one of claims 1 to 93 for use in a diagnostic method performed in a human or animal body. 97. A material according to any of claims 1 to 94 for use in a method of treatment performed in the human or animal body. 98. A material according to any of the preceding claims for use in a surgical method performed on a human or animal body. 99. The material according to any one of claims 1 to 96 for use in a method for producing a biohybrid organ in vivo. 100. The material according to any one of claims 1 to 97, as a carrier for delivering the drug to a human or animal body as needed. 101. The material according to any one of the preceding claims for use in a method for controlling cell growth and / or cell proliferation and / or cell differentiation in a living body. 102. The material according to any one of the preceding claims for use in a method for separating and / or isolating biological material in a living body. 103. A composition comprising a material according to any of the preceding claims and a physiologically acceptable carrier. 104. A pharmaceutical composition comprising a material according to any of the preceding claims 1 to 100 or a composition according to claim 101, a pharmaceutically active ingredient and an optional pharmaceutically active carrier. 105. Use of a material according to any one of claims 1 to 100 or a composition according to claim 101 or a pharmaceutical composition according to claim 102 in a method of treatment performed in a human or animal body. 106. Use of a material according to any of claims 1 to 100 or a composition according to claim 101 or a pharmaceutical composition according to claim 102 in a surgical method performed on a human or animal body. Use. 107. Use of the material according to any one of claims 1 to 100 or the composition according to claim 101 or the pharmaceutical composition according to claim 102 in a diagnostic method performed in a human or animal body. 108. Use of the material according to any one of claims 1 to 100, the composition according to claim 101, or the pharmaceutical composition according to claim 102 in a method for producing a biohybrid organ in vivo. 109. The material according to claim 1 to 100 or the composition according to claim 101, or a composition according to claim 101, as a carrier for delivering the drug to a human or animal body as needed. Use of the pharmaceutical composition according to the above. 110. The material according to any one of claims 1 to 100 or the composition according to claim 101, or 102, in a method for controlling cell growth and / or cell proliferation and / or cell differentiation in a living body. Use of the pharmaceutical composition according to the above. 111. The material according to claim 1 to 100 or the composition according to claim 101 in a method for separating and / or isolating a biological material in a living body.
Use of the pharmaceutical composition according to Item 102. 112. The material according to claim 1 to 100 or the composition according to claim 101 or the composition according to claim 102 in a method for controlling cell growth and / or cell proliferation and / or cell differentiation in vitro. Use of pharmaceutical compositions. 113. A material according to any one of claims 1 to 100 or a composition according to claim 101 or a pharmaceutical composition according to claim 102 in a method for separating and / or isolating a biological material in vitro. Use of things. 114. Use of the material according to claim 1 to 100 or the composition according to claim 101 or the pharmaceutical composition according to claim 102 in a method for producing an in vitro biohybrid organ. 115. Use of a material according to any of claims 1 to 100 or a composition according to claim 101 or a pharmaceutical composition according to claim 102 in the manufacture of an implantable organ or part thereof. . 116. Use of a material according to any of claims 1 to 100 as a carrier for a pharmaceutically active ingredient or a pharmaceutical composition. 117. A method for controlling cell growth and / or proliferation and / or cell differentiation in vitro, said method comprising the step of transforming a cell into a material according to any one of claims 1 to 100 or a composition according to claim 101. 107. An in vitro cell comprising contacting with a substance or the pharmaceutical composition of claim 102, incubating said cells and said material under conditions under which said cells can grow and / or proliferate and / or differentiate. A method for controlling growth and / or proliferation and / or cell differentiation. 118. A method for separating and / or isolating a biomaterial in vitro, wherein the biomaterial to be separated and / or isolated by the method is a material or a material according to any one of claims 1 to 100. 101. contacting the composition of claim 101 or the pharmaceutical composition of claim 102, separating and / or separating the biomaterial and the material.
Alternatively, a method for separating and / or isolating a biomaterial in vitro, which comprises a step of incubating under conditions allowing isolation. 119. A method for producing a biohybrid organ in vitro, comprising the steps of: using the material of any one of claims 1 to 100 or the composition of claim 101; 110. A method for producing a biohybrid organ in vitro, comprising the steps of: contacting with a pharmaceutical composition according to Item 102; and incubating the cells of the biohybrid organ under conditions that allow the production of the biohybrid organ. 120. A method of performing treatment on a human or animal body, said method comprising the step of treating said body with a material according to any of claims 1 to 100 or a composition according to claim 101 or a composition according to claim 101. A method of performing a treatment in a human or animal body comprising the step of contacting the described pharmaceutical composition. 121. A method of performing a surgical procedure on a human or animal body, the method comprising the step of removing the body or material according to any of claims 1 to 100.
103. A method of performing a surgical procedure on a human or animal body, comprising the step of contacting the composition with the composition of claim 102, or the pharmaceutical composition of claim 102. 122. A diagnostic method performed in the human or animal body, said method comprising the step of removing said body from a material according to any of claims 1 to 100 or a composition according to claim 101 or a composition according to claim 101. A diagnostic method carried out in a human or animal body, comprising the step of contacting with the pharmaceutical composition according to 1 above and detecting a signal emitted indirectly or directly by said material. 123. A method for controlling cell growth and / or proliferation and / or cell differentiation in a living body, the method comprising the steps of: (a) preparing a cell according to any one of claims 1 to 100 or a composition according to claim 101; 103. An in vivo cell comprising the steps of contacting with a substance or the pharmaceutical composition of claim 102, and incubating said cells and said material under conditions under which said cells can grow and / or proliferate and / or differentiate. A method for controlling growth and / or proliferation and / or cell differentiation. 124. A method for separating and / or isolating a biological material in a living body, wherein the biological material to be separated and / or isolated by the method is a material or a material according to any one of claims 1 to 100. 101. contacting the composition of claim 101 or the pharmaceutical composition of claim 102, separating and / or separating the biomaterial and the material.
Alternatively, a method of separating and / or isolating a biological material in a living body, comprising a step of incubating under a condition capable of isolation. 125. A method for producing a biohybrid organ in a living body, the method comprising the steps of: producing a cell of the biohybrid organ from the material according to any one of claims 1 to 100 or the composition according to claim 101; Item 110. A method for producing a biohybrid organ in a living body, comprising the steps of: contacting with a pharmaceutical composition according to Item 102; and incubating cells of the biohybrid organ under conditions capable of producing the biohybrid organ. 126. A method of in vivo delivery of a drug to a human or animal body as needed, wherein the method comprises the step of contacting the body with the drug composition of claim 102; A method for in vivo delivery of a drug to a human or animal body as needed, comprising incubating the contacted body under conditions that permit delivery of the drug. 127. The method of making a material according to any of claims 1 to 100, wherein the method comprises: (i) providing a culture substrate having a second contact angle; and (ii) the substrate. Contacting with a composition comprising a plurality of macromolecules. 128. The method of claim 125, wherein said substrate comprises a hydrophobic polymer. 129. The method of claim 125, wherein said substrate is pre-treated before being contacted with said macromolecules. 130. The method of claim 127, wherein said pretreatment is effective in increasing wettability of said substrate. 131. The method of claim 125, wherein said macromolecule comprises a hydrophilic polymer. 132. The macromolecule comprises a potentially reactive polymer.
The method described in. 133. The macromolecule has a molecular weight greater than 400 Da.
The method described in 5. 134. The method of claim 125, wherein said macromolecule comprises a crosslinkable head group, a linker group, a polymer chain, and a functional end group. 135. The method of claim 132, wherein said crosslinkable head group is a photoreactive allyl azide head group. 136. The method of claim 132, wherein said macromolecule further comprises a modifying agent. 137. The method of claim 134, wherein said modifying agent is capable of contacting said substrate and forming a self-assembled monolayer. 138. Claim for producing the material according to claims 1 to 100
125. The method of claims 125-135, further comprising contacting said material with a first determinant comprising a bioactive compound. 139. The bioactive compound is a polypeptide, antibody, polyclonal antibody, monoclonal antibody, immunogenic determinant, antigenic determinant, receptor, receptor binding protein, interleukin, cytokine, cell differentiation factor, cell growth 139. The method of claim 136, wherein the method is selected from the group consisting of a factor and a receptor antagonist. 140. The method of claim 136, wherein said bioactive compound is a membrane-associated and / or extracellular matrix polypeptide autologous to microbial, plant, or mammalian cells. 141. A claim for producing the material according to claims 1 to 100
136. The method according to any of 136 to 138, further comprising contacting the material with a second determinant comprising a biological entity. 142. The method of claim 139, wherein said biological entity comprises a cell or a virus, or a portion thereof. 143. Microbial cells wherein said cells or portions thereof are mammalian cells, including human cells and animal cells, plant cells, eukaryotic microbial cells including yeast and fungi,
141. The method of claim 140, wherein the method is selected from the group consisting of prokaryotic cells, including bacteria. 144. The virus or a portion thereof comprises a mammalian virus including human and animal viruses, a plant virus, a microbial virus including eukaryotic microbial viruses including yeast viruses and fungal viruses, a prokaryotic microbial virus including bacteriophage. 141. The method of claim 140, wherein the method is selected from a group. 145. The method of claim 139, wherein said biological entity comprises a polypeptide or a portion thereof, a nucleic acid portion, a carbohydrate portion, a lipid portion, and complexes thereof. 146. The biological entity wherein the biological entity is a polypeptide, antibody, polyclonal antibody, monoclonal antibody, immunogenic determinant, antigenic determinant, receptor, receptor binding protein, interleukin, cytokine, cell differentiation factor, cell growth factor. 140. The method of claim 139, wherein the method is selected from the group consisting of: and a receptor antagonist.