CA2197000C - Surface-modifying copolymers having cell adhesion properties - Google Patents
Surface-modifying copolymers having cell adhesion properties Download PDFInfo
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- CA2197000C CA2197000C CA002197000A CA2197000A CA2197000C CA 2197000 C CA2197000 C CA 2197000C CA 002197000 A CA002197000 A CA 002197000A CA 2197000 A CA2197000 A CA 2197000A CA 2197000 C CA2197000 C CA 2197000C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
- A61L33/0076—Chemical modification of the substrate
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
- C08G77/42—Block-or graft-polymers containing polysiloxane sequences
- C08G77/458—Block-or graft-polymers containing polysiloxane sequences containing polyurethane sequences
Abstract
A hemocompatible surface-modifying additive is provided for modifying polyurethane or polyurethane-urea substrates. The additive has a polyurethan e or polyurethane urea hard block or an alternative block which is miscible wi th the poly(urethane) or poly(urethane-urea) base polymer, a polysiloxane hydrophobic soft block, an optional hydrophilic spacer and a peptide selecte d from the group consisting of Arg-Gly-Asp, X-Arg-Gly-Asp, Arg-Gly-Asp-X and X - Arg-Gly-Asp-X', wherein X and X' are amino acids.
Description
WO 96140187 PCTlUS96/07500 2i9'~OQO
STIRFACE-MODT_FYT_NG COPOLYMERS
HAVING CELL ADHESION PROPERTIES
Field of the Invention The present invention is directed to novel block copolymers which are hemocompatible surface-modifying additives for modifying polyurethane, poly (urethane urea), polystyrene or polystyrene-containing elastomer substrates.
Backcrround of the Invention Cell, adhesion to natural or synthetic substrates is mediated by the interaction of cell adhesion proteins which correspondingly accommodate cell surface receptors. Cell adhesion proteins are exemplified by fibronectin, vibronectin, collagen, thrombospondin, von Willebrand factor and laminin, all found in the extracellular matrix. These call adhesion proteins have cell surface receptors having varying specificity depending on the particular receptor.
In many cases the cell binding sequences-within the cell adhesion proteins which comprise part of the receptor consist of variants of the sequence RGDX (where x is a variant amino acid)--(RGDX equals Arg-Gly-Asp-X). For example, RGDS is one of the sequences found in fibronectin, fibrinogen and von Willebrand factor which aids adhesion to cells. Fibronectin also contains RGDX-like adhesion sequences such as REDV (Arg-Glu-Asp-Val). Since the sequences within the cell adhesion proteins appear to-be responsible for cell adhesion, there have been-studies of ' the binding capacity of these particular short sequences when -they are not within the context of the larger protein.
For example, J.A. Hubbell et al., Hio/Technology,-1991, ~, 568-571, and Annals of the NY Academy of Sciences, 665, 1992, 253-258, disclosed a study of cell and blood platelet 219'~~~~
binding activity onto surfaces with covalently attached RGD
amino acid sequences: These sequences-supported the spreading and growth of human fibroblasts, human vascular ' smooth muscle cells and human umbilical cord endothelial cells, but did not support the spreading of blood platelets.
Sequences containing REDV attached in the same manner specifically supported the-.spreading and growth of human umbilical cord endothelial cells but also did not support the spreading o~ blood platelets.
Accordingly, there is a need to develop synthetic biomaterials for applications in the human cardiovascular system which will resist blood coagulation by promoting the growth of protective endothelial layers over the surfaces but yet which wouldalso mediate the buildup of thrombus.
Biomaterial surfaces comprising either hydrophilic (e.g., polyethylene oxide) or extremely hydrophobic (e. g., polydimethylsiloxane)are known in-medical devices and are-the subject ofongoing investigation for such cardiovascular applications, but such biomaterials are xiot as thromboresistant as endothelialized surfaces. Thus while it is currently possible to produce devices for relatively short-term functions while using anticoagulants to aid in preventing clotting, there are no suitable materials for lifetime applications.
The present invention provides nonpolar polydimethylsiloxane blocks incorporated into a block copolymer onto which is covalently linked a particular chemical sequence for.increased cell adhesion. The nonpolar polydimethylsiloxane blocks selectively segregate toward the air interface of the substrate polymer surface as a result of the thermodynamic driving force-to minimize surface .free energy. The cell-adhesion-sequence covalently linked , thereto will also be pulled toward the surface of the ' polymer films by virtue of the fact that they are covalently bound to the polydimethylsiloxane. The copolymers can ~. either be blended with substrate polymers in low amounts, coated onto substrate polymers in low amounts, or covalently attached to the substrate polymers. When placed in contact with blood, the cell adhesion amino acid sequences will rise to the surface in the hydrophilic environment, and selectively bind endothelial cells and will not bind blood platelets. Thus endothelial tissue unique to the individual in which the device is implanted will grow over the biomaterial surface and blood will not coagulate on this surface. Since only the surface of the biomaterial will expose the polydimethylsiloxane-amino acid copolymers, a variety of bulk polymeric substrates may be used which have a wide range of mechanical and morphological properties.
arv of-the Inv The copolymers of the- present invention will comprise at least-three blocks or segments, each with a specific function, that is, an anchor block, a surface active block and the amino acid sequence. There may be an optional short hydrophilic spacer between the hydrophobic polydimethylsiloxane surface active block and the amino acid sequence. The function of the anchor block will be to provide the ability to form a film so that the copolymer will not be extracted from the biomaterial surface in the presence of blood, and to provide compatibility with the bulk material. This is an important feature in view of regulatory concerns. The anchor block must be chemically compatible with the bulk of the biomaterial substrate. For example, copolymers with polystyrene anchor blocks will be suitable for blending with or coating onto golystyrene, polystyrene containing elastomers, or polyphenylene oxide.
_ g _ Copolymers containing either polyurethane or polyurea segments would be suitable for blending with polyurethane, polyurea, or polyurethane-urea) bulk materials. Alternatively, a short, hydrophilic, spacer such as poly(2-ethyloxazoline) or polyethylene oxide) can be inserted between the polydimethylsiloxane and the amino acid sequence.
According to one aspect, the invention provides a hemocompatible surface-modifying copolymer additive for modifying polyurethane) or polyurethane urea) substrates, said additive having the formulas An~B~ C -D~ v~ ~A- B~B~ C D~ v~
m P, m P, ~B~ C D v mAn CB -A~B--~ C D~ vJ
P or m P
wherein A is a polyurethane, polyurethane urea) or polystyrene hard block polymer; B is a polysiloxane hydrophobic soft block;
C is. an optional hydrophilic spacer; D is a peptide selected from the group consisting of the sequence -Arg-Gly-Asp and -X-Arg-Gly-Asp, wherein X is an amino acid; and n, v, m and p are each independently an integer greater than 0 and up to 100.
Brief Description of the Drawing Description of the Preferred Embodiments The copolymers provided in accordance with the present invention comprise at least three blocks or segments.
By the term block it is meant that there is one type of recurring unit. Although the term block is a generic term, the term "segment" is sometimes used in the art to represent a relatively short length of repeating units, e.g., less than about ten monomeric units, but preferably less than 3 monomeric units, typically alternating more than once with structural formulas such as ABAB. Block copolymers or segmented copolymers are composed of at least two blocks, one block composed of one type of recurring unit and at least one other block composed of a different type of recurring unit. Block copolymers as defined herein may be linear, cyclic or branched (cross-linked) structures. The preferred block copolymers in accordance with the present invention are linear.
4a i :-The block copolymers according to the present invention are hemo-compatible surface-modifying copolymer additives having the formulas:
-/Y,TJI I A~~ ~ wJ~ITl~
~IB'..E~.p~A~~r o~ I~~A~ B'{ ~~~~~~~.
wherein A is a poly(urethane), polyurethane-urea), or polystyrene hard block; B is a polysiloxane hydrophobic soft-block; C is an optional hydrophilic spacer; and D is peptide sequence.selected from the group consisting of.
Arg-Gly-Asp, X-Arg-Gly-Asp, Arg-GIy-Asp-X and X'-Arg-Gly-Asp-X wherein X and X' are any amino acid; and each n, v, m, and p is independently an integer greater than 0 up to about 5D0. The preferred amino acid for X and X' is Gly.
The hard block (A) need not be a polyurethane) or polyurethane urea), as long as it is a hard block which is thermodynamically miscible with a poly(urethane), or poly (urethane urea) base polymer. Likewise, the hard block need not be a Polystyrene, as long as it is a hard block which is miscible with a polystyrene or polystyrene-containing base polymer.
Copolymers containing the anchor.block and the surface-active block can be made in a variety of ways. For example, if the anchor block is polystyrene an activated terminus polystyrene may be prepared and then reacted with a cyclic siloxane to produce a copolymer of the anchor block (polystyrene)-and the surface active block (polysiloxane).
The copolymer can then be reacted with an amino acid n sequence.
An exemplary-scheme is shown below. In scheme 1 sec-butyl-lithium is reacted with styrene to produce an WO 96/40187 '~ PCT/US96/07500 alkyl lithium terminated polystyrene. This in turn is reacted with cyclic polysiloxane then capped with a silane having an activated terminal (in this case, chloromethyl) ' for further reaction.
w~Qr-iJ r ...
eyaloho anNTHF'°°~a rm. bmp.
.x s x - y ~ ~~i r sohomo 1~ preparation o! Foly(atyrono-co-dimathylsiloxana) ~ s .
In scheme 2 the terminal chloromethyl group is converted to an iodomethyl group then treated with the peptide.
~ Hal' t HaC) fi'N~~ROD
~RaD . ply.arp.p~y.atp schemo 2e preparation of poly(atyrane-oo-dimathylailoxane-co-ammo aoid sequence) 2~.9'~t~Ofl~~~ r Alternatively, the polystyrene polysiloxane copolymer can be end-capped with activated ethyloxazoline groups for reaction with the peptide. Referring below to ' scheme 3, the methylene chloride-terminated polystyrene polysiloxane copolymer is treated with ethyloxazoline to make the ethyloxazoline-terminated intermediates, which after treatment with the peptide GRGD, results in the peptide-terminated copolymer.
~ee~Dr oJ' a~...y r at, 'e m oNorobon:eno vAytox~totlna 1t1'0 !.
H~-GRaD
scheme 7: propagation o! Faly(etyrene-oo-dimethylsiloxene-oo-ethyloxaaoline-oo-ammo aoid seQUenca) - g _ WO 96!40187 i ~ Alternatively, hydroxy functional dimethylsiloxane and-blocking reagents may be prepared whereby the molecular weight of polydimethylsiloxane oligomers can be controlled by synthetic conditions. Thus polysiloxane-urethane blocked .polymers may be prepared modified with appended peptide moieties (such as RGD) which are covalently linked to the polysiloxane portion of the chain via an arginine N-terminal.
A preferred group of hydroxy-terminated oligomeric precursors is described below in scheme 4. (See U.S. Patent 4,689,383 to J.S. Riffle & R.S. Ward, 1987.) .rr . ert,...pw ~N,noy ~--e-toK7r -e- -t~ur-~-air ~~
ona.hnr awr mr. ~.!..c w .pe.r . r.a r.onuil v. a.erua hr ..~.
_a-teHr~- o- -iayr-e-ow u) i~r .w eo-ars,-o-~~ ~ ~oyy-~o-u4 -.orr w ~eawm wr.wur wry r.kwr~snr H4nwl Scheme 4: Preparation of hydroxyfunctional dimethylsiloxane endblocking reagent (I), and controlled molecular weight polydimethylsiloxane oligomers with hydroxyl endgroups.
g -PCT/US96/07500~
A polydimethylsiloxane is reacted with an epoxy allyl ether to form the epoxy-terminated siloxane. The epoxy end groups are then opened with methanol and then ' coeguilibration is used to create oligomers of desired molecular weight ranges us~.ng D4, a cyclosiloxane tetramer.
Cyclosiloxanes having functional substituents such as cyanopropyl in addition to methyl attached to the silicon atoms can be polymerized with the D, to prepare analogous eiloxane o-ligomers with pendent functional groups along the oligomer backbone. The yield of the desired polymer is sensitive to the size of the substituent group on the silicon atoms in the siloxane system whereby the yield decreases for hydrogen, alkyl groups, and trifluoromethylpropyl as the subetituents increase in size.
However, a cyanopropyl-methylcyclotetrasiloxane yields a high volume fraction of polymer (about 75% when equilibrated with trifluoromethanesulfonic acid catalyst).
A polyurethane copolymer backbone with pendent benzyl butyrate substituents may be synthesized in a manner similar to the methods used for preparing block copolymers.
Polysiloxane prepolymers may be end-capped by reaction with an excess of methylene-diphenyldiisocyanate and then extended with 1,4-butanediol. The-benzyl esters may be removed by known methods, such as by hydrogenation. This is shown in scheme 5, below.
i 219~00a ~IrOH~_~-d t ft=PtCI~
(lonty! butYratv t:lrbstltuW
wIQ b~ dltl~nltH 's1') ~1. totu~nohQ. btoubonolta !. teluswftrllGo ~etdlnllwc (nmovmntvr) Scheme 5: Siloxane cyclic tetramer with protected carboxylic acid functionality.
The peptides containing the amino acid segments such as RGD, may be prepared by known procedures such as that shown in scheme 6.
SCHE~~SE 6 tBoo~NH-CH:-COON ~ H~-'~'~Hx'Ph ~~C00-CHzPh H:~:
t8oo~~1H-CIUz-CO-Hti-CH-C00-CHrPh ~Hy-CDO-CHaPh ~1y TrlBic Acid 2}TrtvthyJamla~
itZlt-CH=-CO-HH-CH-C00-CH~-Ph ~HZ~C00-CH=Ph HzN-CHi-CO-HH- f H-COO~CHYPh CH?~C00-CHZPh DCCICH=CI=
rti-CH=-CO-HH-GJ'H-COO-CHrPh Clis~COO-CHZPh Scheme 6: RGD segment preparation.
_12_ 7~TritBC acid 2} Trloihytamlne The amine terminus of these graft segments may be deblocked and the grafts bonded to the pendent carboxypropyl units on a block copolymer using dicyclohexyl carbodiimide as an activatingagent as shown in scheme 7.
- SCHEME '7 s tio8 ~loot !4N ~bet DCC/CII~Ch (hrot~ot~d ROD) Nlltd 8.".u; .8 Scheme 7: Attachment to RGD segments tothe polymer backbone.
WO 96/40187 PCTlUS96/07500 i The remaining benzyl esterprotecting groups on the aspartic acid may be deprotected by hydrogenation. Deprotection reactions conducted in the presence of the acid liable siloxane bonds may be achieved using hydrogenation, as opposed to acid hydrolysis, to ensure that the copolymer backbone remains unaffected.
As an alternative to the last step in scheme 4, the following process (shown in scheme 8) may be utilized to prepare hydroxy-functional polysiloxane oligomers with pendent protected carboxy groups.
_ i~' ,_ -°w c~4 -°~
"° ; H-o~y-°-~c~°'--i~",~.O.o~4_~
/vtrtse cola, nut v ~»d oyotlo~
Scheme 8: Preparation of hydroxy functional polysiloxane r oligomers with pendent protected carboxy groups. Due to the mechanism of equilibration reactions, the sequence of ~~x~~.
and ~~y~~ units will be randomly distributed along the chain_ The sequences of the repeat units indicated with subscripts x and y are randomly distributed along the chain.
Polyethylene oxide spacers can be added to the amine terminal of the arginine residue via the method outlined in schemes 9 and 11 using protein synthesis techniques (Merrifield method) to produce - PEO-RGD pendent groups.
c. nmnw~r~ n OzN ~0 0 + K + H3C CHz 1. -80°C.
Z~THEN SUCCINIC ANHYDRIDE
OzN
0-~-CHz CHZ O~C (CHz)z COOH
Hz/ Pd HzN
0 CCHz CHz ~X C (CHz)z COOH
Scheme 9: Preparation of polyethylene oxide spacers with an amine terminus and with a carboxylic acid terminus. The starting material is preferably the p-vitro-phenoxide salt.
W O 96/40187 PG°f/US96I07500 2I970~0 SCHEME 1, ~VNo~L~ooN .lf~t( ,.~.~~' _pp~r f ~trtW aoW
~trlslAylmdM
Scheme 11: Preparation of RGD segments with hydrophilic PEO
spacer units.
This oligomeric group may be appended to the carboxyl group on the siloxane portion of the copolymer by coupling the amine terminus to the PEO unit as shown in scheme 10.
'~~~~DDO
~-~CHPHPL~<a~-~ -~~n,~ ~ ~ocn,~n, u.~rHC°o~
Scheme 10: Polysiloxane-urethane block copolymers containing pendent amino acids appended via hydrophilic spacers.
It will be realized that other types of spacers may be utilized such as poly(aolkyloxazoline), polyacrylamides, polylvinylpyrrolidone). However, the polyethylene oxide spacers or other polyalkylene oxide spacers are preferred since the binding strength to hydrogen bonding surfaces of l0 ether oxygens is expected to be less than the binding strength of the amino bonds of the amino acid segments.
Therefore, the polyethylene oxide spacers would not substantially interfere with the binding process of the amino acids to the cell surfaces.
ii ADC 'cn,Cl, W O 96140187 CA 0 219 7 0 0 0 2 0 0 0 - 0 3 -17 p~~gg6107500 Referring to Scheme 5, vinylacetic acid can either be coupled directly to benzylalcohol using known activating agents (e.g. dicyclohexylcarbodiimide), or it can first be converted to the corresponding acid chloride and subsequently esterified to form benzyl vinylacetate, and then is reacted with dichloromethylsilane to produce benzyl butyrate silane. This is then converted to the cyclic siloxane tetramer having protected carboxylic acid functionalities. Referring to Scheme 8, this siloxane tetramer can then be equilibrated with D4 and siloxane hydroxy terminated monomers to form the hydroxy functional terminated polysiloxane oligomers with pendent protected carboxy groups. Referring to Scheme 6 there is shown a convertior~al Merrifield synthesis scheme for coupling amino acids to make the peptide RGD. Referring to Scheme 9 there is shown the preparation of polyethylene oxide spacers having an amine terminus and a carboxylic acid terminus.
Referring to Scheme 7 there is shown the addition of the protected peptides to the carboxy terminal pendant groups of hydroxy functional polysiloxane oligomers and then removal of the protecting groups to form, in this case, a polyurethane polysiloxane polymer backbone having pendent RGD groups. Referring to Scheme 11 there is shown the attachment of the PEO spacers to the amino terminus of the RGD end of the RGD peptide.
It will be realized that other linear polymers may be utilized such as polysiloxane-polylactone block copolymers, examples of which are described in Patent No.
4,663,413. Methods of preparation of dihydroxy functional polydimethylsiloxane-oligomers are further disclosed in Patent No. 4,689,383.
219700a i The polymers according to the present invention are useful for surface modification of base polymers which are n poly(urethane), polyurethane-urea)s , polystyrenes or polystyrene-containing elastomers, to allow endothelial cell adhesion thereto, thereby forming endothelial monolayera which are nonthrombogenic. Typically, the polymers according to the present invention may be utilized as additives to the poly(urethane), polyurethane-urea), polystyrene, or polystyrene-containing elastomer base IO polymer in amounts of 0-.1 to 20 percent by weight. The base polymer modified according-to the present invention may be used in blood-contacting devices, such as vascular prostheses in the venous or arterial system, heart patches, heart valves, as the outer encapsulants of implantable i5 devices such as pacemakers, catheters or the outer sheath of catheters in contact with body fluids, temporary coverings on open wounds. The surface may also be utilized in extracorporeal devices to provide channels-through which body fluids may be passed in heart, lung and kidney 20 machines. -
STIRFACE-MODT_FYT_NG COPOLYMERS
HAVING CELL ADHESION PROPERTIES
Field of the Invention The present invention is directed to novel block copolymers which are hemocompatible surface-modifying additives for modifying polyurethane, poly (urethane urea), polystyrene or polystyrene-containing elastomer substrates.
Backcrround of the Invention Cell, adhesion to natural or synthetic substrates is mediated by the interaction of cell adhesion proteins which correspondingly accommodate cell surface receptors. Cell adhesion proteins are exemplified by fibronectin, vibronectin, collagen, thrombospondin, von Willebrand factor and laminin, all found in the extracellular matrix. These call adhesion proteins have cell surface receptors having varying specificity depending on the particular receptor.
In many cases the cell binding sequences-within the cell adhesion proteins which comprise part of the receptor consist of variants of the sequence RGDX (where x is a variant amino acid)--(RGDX equals Arg-Gly-Asp-X). For example, RGDS is one of the sequences found in fibronectin, fibrinogen and von Willebrand factor which aids adhesion to cells. Fibronectin also contains RGDX-like adhesion sequences such as REDV (Arg-Glu-Asp-Val). Since the sequences within the cell adhesion proteins appear to-be responsible for cell adhesion, there have been-studies of ' the binding capacity of these particular short sequences when -they are not within the context of the larger protein.
For example, J.A. Hubbell et al., Hio/Technology,-1991, ~, 568-571, and Annals of the NY Academy of Sciences, 665, 1992, 253-258, disclosed a study of cell and blood platelet 219'~~~~
binding activity onto surfaces with covalently attached RGD
amino acid sequences: These sequences-supported the spreading and growth of human fibroblasts, human vascular ' smooth muscle cells and human umbilical cord endothelial cells, but did not support the spreading of blood platelets.
Sequences containing REDV attached in the same manner specifically supported the-.spreading and growth of human umbilical cord endothelial cells but also did not support the spreading o~ blood platelets.
Accordingly, there is a need to develop synthetic biomaterials for applications in the human cardiovascular system which will resist blood coagulation by promoting the growth of protective endothelial layers over the surfaces but yet which wouldalso mediate the buildup of thrombus.
Biomaterial surfaces comprising either hydrophilic (e.g., polyethylene oxide) or extremely hydrophobic (e. g., polydimethylsiloxane)are known in-medical devices and are-the subject ofongoing investigation for such cardiovascular applications, but such biomaterials are xiot as thromboresistant as endothelialized surfaces. Thus while it is currently possible to produce devices for relatively short-term functions while using anticoagulants to aid in preventing clotting, there are no suitable materials for lifetime applications.
The present invention provides nonpolar polydimethylsiloxane blocks incorporated into a block copolymer onto which is covalently linked a particular chemical sequence for.increased cell adhesion. The nonpolar polydimethylsiloxane blocks selectively segregate toward the air interface of the substrate polymer surface as a result of the thermodynamic driving force-to minimize surface .free energy. The cell-adhesion-sequence covalently linked , thereto will also be pulled toward the surface of the ' polymer films by virtue of the fact that they are covalently bound to the polydimethylsiloxane. The copolymers can ~. either be blended with substrate polymers in low amounts, coated onto substrate polymers in low amounts, or covalently attached to the substrate polymers. When placed in contact with blood, the cell adhesion amino acid sequences will rise to the surface in the hydrophilic environment, and selectively bind endothelial cells and will not bind blood platelets. Thus endothelial tissue unique to the individual in which the device is implanted will grow over the biomaterial surface and blood will not coagulate on this surface. Since only the surface of the biomaterial will expose the polydimethylsiloxane-amino acid copolymers, a variety of bulk polymeric substrates may be used which have a wide range of mechanical and morphological properties.
arv of-the Inv The copolymers of the- present invention will comprise at least-three blocks or segments, each with a specific function, that is, an anchor block, a surface active block and the amino acid sequence. There may be an optional short hydrophilic spacer between the hydrophobic polydimethylsiloxane surface active block and the amino acid sequence. The function of the anchor block will be to provide the ability to form a film so that the copolymer will not be extracted from the biomaterial surface in the presence of blood, and to provide compatibility with the bulk material. This is an important feature in view of regulatory concerns. The anchor block must be chemically compatible with the bulk of the biomaterial substrate. For example, copolymers with polystyrene anchor blocks will be suitable for blending with or coating onto golystyrene, polystyrene containing elastomers, or polyphenylene oxide.
_ g _ Copolymers containing either polyurethane or polyurea segments would be suitable for blending with polyurethane, polyurea, or polyurethane-urea) bulk materials. Alternatively, a short, hydrophilic, spacer such as poly(2-ethyloxazoline) or polyethylene oxide) can be inserted between the polydimethylsiloxane and the amino acid sequence.
According to one aspect, the invention provides a hemocompatible surface-modifying copolymer additive for modifying polyurethane) or polyurethane urea) substrates, said additive having the formulas An~B~ C -D~ v~ ~A- B~B~ C D~ v~
m P, m P, ~B~ C D v mAn CB -A~B--~ C D~ vJ
P or m P
wherein A is a polyurethane, polyurethane urea) or polystyrene hard block polymer; B is a polysiloxane hydrophobic soft block;
C is. an optional hydrophilic spacer; D is a peptide selected from the group consisting of the sequence -Arg-Gly-Asp and -X-Arg-Gly-Asp, wherein X is an amino acid; and n, v, m and p are each independently an integer greater than 0 and up to 100.
Brief Description of the Drawing Description of the Preferred Embodiments The copolymers provided in accordance with the present invention comprise at least three blocks or segments.
By the term block it is meant that there is one type of recurring unit. Although the term block is a generic term, the term "segment" is sometimes used in the art to represent a relatively short length of repeating units, e.g., less than about ten monomeric units, but preferably less than 3 monomeric units, typically alternating more than once with structural formulas such as ABAB. Block copolymers or segmented copolymers are composed of at least two blocks, one block composed of one type of recurring unit and at least one other block composed of a different type of recurring unit. Block copolymers as defined herein may be linear, cyclic or branched (cross-linked) structures. The preferred block copolymers in accordance with the present invention are linear.
4a i :-The block copolymers according to the present invention are hemo-compatible surface-modifying copolymer additives having the formulas:
-/Y,TJI I A~~ ~ wJ~ITl~
~IB'..E~.p~A~~r o~ I~~A~ B'{ ~~~~~~~.
wherein A is a poly(urethane), polyurethane-urea), or polystyrene hard block; B is a polysiloxane hydrophobic soft-block; C is an optional hydrophilic spacer; and D is peptide sequence.selected from the group consisting of.
Arg-Gly-Asp, X-Arg-Gly-Asp, Arg-GIy-Asp-X and X'-Arg-Gly-Asp-X wherein X and X' are any amino acid; and each n, v, m, and p is independently an integer greater than 0 up to about 5D0. The preferred amino acid for X and X' is Gly.
The hard block (A) need not be a polyurethane) or polyurethane urea), as long as it is a hard block which is thermodynamically miscible with a poly(urethane), or poly (urethane urea) base polymer. Likewise, the hard block need not be a Polystyrene, as long as it is a hard block which is miscible with a polystyrene or polystyrene-containing base polymer.
Copolymers containing the anchor.block and the surface-active block can be made in a variety of ways. For example, if the anchor block is polystyrene an activated terminus polystyrene may be prepared and then reacted with a cyclic siloxane to produce a copolymer of the anchor block (polystyrene)-and the surface active block (polysiloxane).
The copolymer can then be reacted with an amino acid n sequence.
An exemplary-scheme is shown below. In scheme 1 sec-butyl-lithium is reacted with styrene to produce an WO 96/40187 '~ PCT/US96/07500 alkyl lithium terminated polystyrene. This in turn is reacted with cyclic polysiloxane then capped with a silane having an activated terminal (in this case, chloromethyl) ' for further reaction.
w~Qr-iJ r ...
eyaloho anNTHF'°°~a rm. bmp.
.x s x - y ~ ~~i r sohomo 1~ preparation o! Foly(atyrono-co-dimathylsiloxana) ~ s .
In scheme 2 the terminal chloromethyl group is converted to an iodomethyl group then treated with the peptide.
~ Hal' t HaC) fi'N~~ROD
~RaD . ply.arp.p~y.atp schemo 2e preparation of poly(atyrane-oo-dimathylailoxane-co-ammo aoid sequence) 2~.9'~t~Ofl~~~ r Alternatively, the polystyrene polysiloxane copolymer can be end-capped with activated ethyloxazoline groups for reaction with the peptide. Referring below to ' scheme 3, the methylene chloride-terminated polystyrene polysiloxane copolymer is treated with ethyloxazoline to make the ethyloxazoline-terminated intermediates, which after treatment with the peptide GRGD, results in the peptide-terminated copolymer.
~ee~Dr oJ' a~...y r at, 'e m oNorobon:eno vAytox~totlna 1t1'0 !.
H~-GRaD
scheme 7: propagation o! Faly(etyrene-oo-dimethylsiloxene-oo-ethyloxaaoline-oo-ammo aoid seQUenca) - g _ WO 96!40187 i ~ Alternatively, hydroxy functional dimethylsiloxane and-blocking reagents may be prepared whereby the molecular weight of polydimethylsiloxane oligomers can be controlled by synthetic conditions. Thus polysiloxane-urethane blocked .polymers may be prepared modified with appended peptide moieties (such as RGD) which are covalently linked to the polysiloxane portion of the chain via an arginine N-terminal.
A preferred group of hydroxy-terminated oligomeric precursors is described below in scheme 4. (See U.S. Patent 4,689,383 to J.S. Riffle & R.S. Ward, 1987.) .rr . ert,...pw ~N,noy ~--e-toK7r -e- -t~ur-~-air ~~
ona.hnr awr mr. ~.!..c w .pe.r . r.a r.onuil v. a.erua hr ..~.
_a-teHr~- o- -iayr-e-ow u) i~r .w eo-ars,-o-~~ ~ ~oyy-~o-u4 -.orr w ~eawm wr.wur wry r.kwr~snr H4nwl Scheme 4: Preparation of hydroxyfunctional dimethylsiloxane endblocking reagent (I), and controlled molecular weight polydimethylsiloxane oligomers with hydroxyl endgroups.
g -PCT/US96/07500~
A polydimethylsiloxane is reacted with an epoxy allyl ether to form the epoxy-terminated siloxane. The epoxy end groups are then opened with methanol and then ' coeguilibration is used to create oligomers of desired molecular weight ranges us~.ng D4, a cyclosiloxane tetramer.
Cyclosiloxanes having functional substituents such as cyanopropyl in addition to methyl attached to the silicon atoms can be polymerized with the D, to prepare analogous eiloxane o-ligomers with pendent functional groups along the oligomer backbone. The yield of the desired polymer is sensitive to the size of the substituent group on the silicon atoms in the siloxane system whereby the yield decreases for hydrogen, alkyl groups, and trifluoromethylpropyl as the subetituents increase in size.
However, a cyanopropyl-methylcyclotetrasiloxane yields a high volume fraction of polymer (about 75% when equilibrated with trifluoromethanesulfonic acid catalyst).
A polyurethane copolymer backbone with pendent benzyl butyrate substituents may be synthesized in a manner similar to the methods used for preparing block copolymers.
Polysiloxane prepolymers may be end-capped by reaction with an excess of methylene-diphenyldiisocyanate and then extended with 1,4-butanediol. The-benzyl esters may be removed by known methods, such as by hydrogenation. This is shown in scheme 5, below.
i 219~00a ~IrOH~_~-d t ft=PtCI~
(lonty! butYratv t:lrbstltuW
wIQ b~ dltl~nltH 's1') ~1. totu~nohQ. btoubonolta !. teluswftrllGo ~etdlnllwc (nmovmntvr) Scheme 5: Siloxane cyclic tetramer with protected carboxylic acid functionality.
The peptides containing the amino acid segments such as RGD, may be prepared by known procedures such as that shown in scheme 6.
SCHE~~SE 6 tBoo~NH-CH:-COON ~ H~-'~'~Hx'Ph ~~C00-CHzPh H:~:
t8oo~~1H-CIUz-CO-Hti-CH-C00-CHrPh ~Hy-CDO-CHaPh ~1y TrlBic Acid 2}TrtvthyJamla~
itZlt-CH=-CO-HH-CH-C00-CH~-Ph ~HZ~C00-CH=Ph HzN-CHi-CO-HH- f H-COO~CHYPh CH?~C00-CHZPh DCCICH=CI=
rti-CH=-CO-HH-GJ'H-COO-CHrPh Clis~COO-CHZPh Scheme 6: RGD segment preparation.
_12_ 7~TritBC acid 2} Trloihytamlne The amine terminus of these graft segments may be deblocked and the grafts bonded to the pendent carboxypropyl units on a block copolymer using dicyclohexyl carbodiimide as an activatingagent as shown in scheme 7.
- SCHEME '7 s tio8 ~loot !4N ~bet DCC/CII~Ch (hrot~ot~d ROD) Nlltd 8.".u; .8 Scheme 7: Attachment to RGD segments tothe polymer backbone.
WO 96/40187 PCTlUS96/07500 i The remaining benzyl esterprotecting groups on the aspartic acid may be deprotected by hydrogenation. Deprotection reactions conducted in the presence of the acid liable siloxane bonds may be achieved using hydrogenation, as opposed to acid hydrolysis, to ensure that the copolymer backbone remains unaffected.
As an alternative to the last step in scheme 4, the following process (shown in scheme 8) may be utilized to prepare hydroxy-functional polysiloxane oligomers with pendent protected carboxy groups.
_ i~' ,_ -°w c~4 -°~
"° ; H-o~y-°-~c~°'--i~",~.O.o~4_~
/vtrtse cola, nut v ~»d oyotlo~
Scheme 8: Preparation of hydroxy functional polysiloxane r oligomers with pendent protected carboxy groups. Due to the mechanism of equilibration reactions, the sequence of ~~x~~.
and ~~y~~ units will be randomly distributed along the chain_ The sequences of the repeat units indicated with subscripts x and y are randomly distributed along the chain.
Polyethylene oxide spacers can be added to the amine terminal of the arginine residue via the method outlined in schemes 9 and 11 using protein synthesis techniques (Merrifield method) to produce - PEO-RGD pendent groups.
c. nmnw~r~ n OzN ~0 0 + K + H3C CHz 1. -80°C.
Z~THEN SUCCINIC ANHYDRIDE
OzN
0-~-CHz CHZ O~C (CHz)z COOH
Hz/ Pd HzN
0 CCHz CHz ~X C (CHz)z COOH
Scheme 9: Preparation of polyethylene oxide spacers with an amine terminus and with a carboxylic acid terminus. The starting material is preferably the p-vitro-phenoxide salt.
W O 96/40187 PG°f/US96I07500 2I970~0 SCHEME 1, ~VNo~L~ooN .lf~t( ,.~.~~' _pp~r f ~trtW aoW
~trlslAylmdM
Scheme 11: Preparation of RGD segments with hydrophilic PEO
spacer units.
This oligomeric group may be appended to the carboxyl group on the siloxane portion of the copolymer by coupling the amine terminus to the PEO unit as shown in scheme 10.
'~~~~DDO
~-~CHPHPL~<a~-~ -~~n,~ ~ ~ocn,~n, u.~rHC°o~
Scheme 10: Polysiloxane-urethane block copolymers containing pendent amino acids appended via hydrophilic spacers.
It will be realized that other types of spacers may be utilized such as poly(aolkyloxazoline), polyacrylamides, polylvinylpyrrolidone). However, the polyethylene oxide spacers or other polyalkylene oxide spacers are preferred since the binding strength to hydrogen bonding surfaces of l0 ether oxygens is expected to be less than the binding strength of the amino bonds of the amino acid segments.
Therefore, the polyethylene oxide spacers would not substantially interfere with the binding process of the amino acids to the cell surfaces.
ii ADC 'cn,Cl, W O 96140187 CA 0 219 7 0 0 0 2 0 0 0 - 0 3 -17 p~~gg6107500 Referring to Scheme 5, vinylacetic acid can either be coupled directly to benzylalcohol using known activating agents (e.g. dicyclohexylcarbodiimide), or it can first be converted to the corresponding acid chloride and subsequently esterified to form benzyl vinylacetate, and then is reacted with dichloromethylsilane to produce benzyl butyrate silane. This is then converted to the cyclic siloxane tetramer having protected carboxylic acid functionalities. Referring to Scheme 8, this siloxane tetramer can then be equilibrated with D4 and siloxane hydroxy terminated monomers to form the hydroxy functional terminated polysiloxane oligomers with pendent protected carboxy groups. Referring to Scheme 6 there is shown a convertior~al Merrifield synthesis scheme for coupling amino acids to make the peptide RGD. Referring to Scheme 9 there is shown the preparation of polyethylene oxide spacers having an amine terminus and a carboxylic acid terminus.
Referring to Scheme 7 there is shown the addition of the protected peptides to the carboxy terminal pendant groups of hydroxy functional polysiloxane oligomers and then removal of the protecting groups to form, in this case, a polyurethane polysiloxane polymer backbone having pendent RGD groups. Referring to Scheme 11 there is shown the attachment of the PEO spacers to the amino terminus of the RGD end of the RGD peptide.
It will be realized that other linear polymers may be utilized such as polysiloxane-polylactone block copolymers, examples of which are described in Patent No.
4,663,413. Methods of preparation of dihydroxy functional polydimethylsiloxane-oligomers are further disclosed in Patent No. 4,689,383.
219700a i The polymers according to the present invention are useful for surface modification of base polymers which are n poly(urethane), polyurethane-urea)s , polystyrenes or polystyrene-containing elastomers, to allow endothelial cell adhesion thereto, thereby forming endothelial monolayera which are nonthrombogenic. Typically, the polymers according to the present invention may be utilized as additives to the poly(urethane), polyurethane-urea), polystyrene, or polystyrene-containing elastomer base IO polymer in amounts of 0-.1 to 20 percent by weight. The base polymer modified according-to the present invention may be used in blood-contacting devices, such as vascular prostheses in the venous or arterial system, heart patches, heart valves, as the outer encapsulants of implantable i5 devices such as pacemakers, catheters or the outer sheath of catheters in contact with body fluids, temporary coverings on open wounds. The surface may also be utilized in extracorporeal devices to provide channels-through which body fluids may be passed in heart, lung and kidney 20 machines. -
Claims (11)
1. A hemocompatible surface-modifying copolymer additive for modifying poly(urethane) or poly(urethane urea) substrates, said additive having the formulas wherein A is a polyurethane, poly(urethane urea) or polystyrene hard block polymer; B is a polysiloxane hydrophobic soft block;
C is an optional hydrophilic spacer; D is a peptide selected from the group consisting of the sequence -Arg-Gly-Asp and -X-Arg-Gly-Asp, wherein X is an amino acid; and n, v, m and p are each independently an integer greater than 0 and up to 100.
C is an optional hydrophilic spacer; D is a peptide selected from the group consisting of the sequence -Arg-Gly-Asp and -X-Arg-Gly-Asp, wherein X is an amino acid; and n, v, m and p are each independently an integer greater than 0 and up to 100.
2. ~An additive according to Claim 1 wherein X is Gly.
3. ~An additive according to Claim 1 wherein said spacer is a polyalkylene oxide.
4. ~An additive according to Claim 1 wherein said additive is of the formula
5. ~An additive according to Claim 1 wherein said additive is of the formula
6. An additive according to Claim 1 wherein said additive is of the formula
7. An additive according to Claim 1 wherein said additive is of the formula
8. A blood-contacting device comprising a blood-contacting surface of a base polymer and an additive according to claim 1 of the formula:
or mixtures of two or more different additives of said formulas.
or mixtures of two or more different additives of said formulas.
9. A method of modifying a poly(urethane) substrate to impart a hemocompatible surface thereto, comprising the step of incorporating into said substrate an effective hemocompatible surface-active amount of an additive according to Claim 1 of the formula:
or mixtures of two or more different additives of said formulas.
or mixtures of two or more different additives of said formulas.
10. A method of modifying a poly(urethane) substrate to impart a hemocompatible surface thereto, comprising the step of incorporating into said substrate an effective hemocompatible surface-active amount of an additive according to any one of Claims 1 through 7.
11. A method of modifying a polyurethane urea) substrate to impart a hemocompatible surface thereto, comprising the step of incorporating into said substrate an effective hemocompatible surface-active amount of an additive according to any one of Claims 1 through 7.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/487,604 | 1995-06-07 | ||
| US08/487,604 US5733538A (en) | 1995-06-07 | 1995-06-07 | Surface-modifying copolymers having cell adhesion properties |
| PCT/US1996/007500 WO1996040187A1 (en) | 1995-06-07 | 1996-05-22 | Surface-modifying copolymers having cell adhesion properties |
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|---|---|
| CA2197000A1 CA2197000A1 (en) | 1996-12-19 |
| CA2197000C true CA2197000C (en) | 2003-10-21 |
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|---|---|---|---|
| CA002197000A Expired - Fee Related CA2197000C (en) | 1995-06-07 | 1996-05-22 | Surface-modifying copolymers having cell adhesion properties |
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|---|---|
| US (1) | US5733538A (en) |
| EP (1) | EP0779815B1 (en) |
| JP (1) | JP2001516228A (en) |
| AU (1) | AU5802596A (en) |
| CA (1) | CA2197000C (en) |
| DE (1) | DE69612267T2 (en) |
| ES (1) | ES2155188T3 (en) |
| WO (1) | WO1996040187A1 (en) |
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| SG66458A1 (en) * | 1997-05-15 | 1999-07-20 | Givaudan Roure Int | Fragrance precursor compounds |
| US6548263B1 (en) | 1997-05-29 | 2003-04-15 | Cellomics, Inc. | Miniaturized cell array methods and apparatus for cell-based screening |
| US6136872A (en) * | 1998-11-20 | 2000-10-24 | Shell Oil Company | Freeze-dried polystyrene-polysiloxane foams |
| AUPQ100699A0 (en) | 1999-06-17 | 1999-07-08 | Northern Sydney Area Health Service | An assist device for the failing heart |
| US6403063B1 (en) | 1999-07-26 | 2002-06-11 | Kenneth I. Sawyer | Method of treating nail fungus |
| KR100312178B1 (en) * | 1999-10-06 | 2001-11-03 | 서경배 | the new amino acid silicon polymers, the method for producing them, the amino acid silicon polymer-suface treated cosmetic particles, and the cosmetic composition including the above particles |
| AU2001294671A1 (en) | 2000-09-25 | 2002-04-08 | The Board Of Trustees Of The University Of Illinois | Microfabrication of membranes for the growth of cells |
| AUPR333301A0 (en) * | 2001-02-23 | 2001-03-22 | Northern Sydney Area Health Service | Determining the volume of a normal heart and its pathological and treated variants by using dimension sensors |
| US6783709B2 (en) * | 2002-07-10 | 2004-08-31 | The Regents Of The University Of California | Self-healing organosiloxane materials containing reversible and energy-dispersive crosslinking domains |
| ATE459380T1 (en) * | 2002-08-13 | 2010-03-15 | Medtronic Inc | DOSAGE SYSTEMS OF ACTIVE INGREDIENTS WITH POLY(ETHYLENE-CO(METH)ACRYLATES, MEDICAL DEVICE AND METHOD |
| CA2494188A1 (en) * | 2002-08-13 | 2004-02-19 | Medtronic, Inc. | Active agent delivery system including a hydrophobic cellulose derivative |
| AU2003258209A1 (en) * | 2002-08-13 | 2004-02-25 | Medtronic, Inc. | Active agent delivery systems, medical devices, and methods |
| US20040033251A1 (en) * | 2002-08-13 | 2004-02-19 | Medtronic, Inc. | Active agent delivery system including a polyurethane, medical device, and method |
| AU2003265446A1 (en) * | 2002-08-13 | 2004-02-25 | Medtronic, Inc. | Active agent delivery system including a hydrophilic polymer, medical device, and method |
| DE60333566D1 (en) * | 2002-08-13 | 2010-09-09 | Medtronic Inc | MEDICAL DEVICE WITH IMPROVED LIABILITY BETWEEN A POLYMERIC TOUCH AND A SUBSTRATE |
| GB0222522D0 (en) | 2002-09-27 | 2002-11-06 | Controlled Therapeutics Sct | Water-swellable polymers |
| CA2535345A1 (en) * | 2003-08-13 | 2005-03-03 | Medtronic, Inc. | Active agent delivery systems including a miscible polymer blend, medical devices, and methods |
| US20050095218A1 (en) * | 2003-10-29 | 2005-05-05 | The Procter & Gamble Company | Personal care composition containing a detersive surfactant, an antidandruff component, and ketoamide surfactants |
| WO2005112974A2 (en) * | 2004-05-14 | 2005-12-01 | William Marsh Rice University | Peptide-modified polyurethane compositions and associated methods |
| GB0417401D0 (en) | 2004-08-05 | 2004-09-08 | Controlled Therapeutics Sct | Stabilised prostaglandin composition |
| US8084275B2 (en) * | 2005-02-08 | 2011-12-27 | Fujifilm Corporation | Magnetic composite body, production method thereof, method for removing substance with mannose on its surface, and method for concentrating substance with mannose on its surface |
| JP2007040978A (en) * | 2005-06-30 | 2007-02-15 | Fujifilm Corp | Method for separating objective element using magnetic nanoparticles |
| EP2213293A3 (en) * | 2005-12-08 | 2011-09-28 | The Polymer Technology Group Incorporated | Self-assembling monomers and oligomers as surface-modifying endgroups for polymers |
| GB0613333D0 (en) | 2006-07-05 | 2006-08-16 | Controlled Therapeutics Sct | Hydrophilic polyurethane compositions |
| GB0613638D0 (en) | 2006-07-08 | 2006-08-16 | Controlled Therapeutics Sct | Polyurethane elastomers |
| GB0620685D0 (en) * | 2006-10-18 | 2006-11-29 | Controlled Therapeutics Sct | Bioresorbable polymers |
| JP5062820B2 (en) * | 2007-03-23 | 2012-10-31 | 株式会社Ａｄｅｋａ | Cyclic siloxane compound and positive resist composition using the same |
| US9994812B2 (en) | 2012-04-04 | 2018-06-12 | University Of Washington Through Its Center For Commercialization | Systems and method for engineering muscle tissue |
| WO2014065017A1 (en) * | 2012-10-26 | 2014-05-01 | 独立行政法人国立循環器病研究センター | Artificial blood vessel, and method for producing artificial blood vessel |
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| US4689383A (en) * | 1986-03-18 | 1987-08-25 | Thoratec Laboratories Corp. | Hydroxyl-functional disiloxanes and polysiloxane oligomers |
| US5079600A (en) * | 1987-03-06 | 1992-01-07 | Schnur Joel M | High resolution patterning on solid substrates |
| US4789601A (en) * | 1987-05-04 | 1988-12-06 | Banes Albert J | Biocompatible polyorganosiloxane composition for cell culture apparatus |
| US4839280A (en) * | 1987-05-04 | 1989-06-13 | Banes Albert J | Apparatus for applying stress to cell cultures |
| US4822741A (en) * | 1987-05-04 | 1989-04-18 | Banes Albert J | Biocompatible polyorganosiloxane composition for cell culture apparatus |
| US5330911A (en) * | 1989-09-28 | 1994-07-19 | Board Of Regents, The University Of Texas System | Surfaces having desirable cell adhesive effects |
| US5369012A (en) * | 1992-03-26 | 1994-11-29 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals |
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1995
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- 1996-05-22 ES ES96914755T patent/ES2155188T3/en not_active Expired - Lifetime
- 1996-05-22 CA CA002197000A patent/CA2197000C/en not_active Expired - Fee Related
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| JP2001516228A (en) | 2001-09-25 |
| EP0779815A4 (en) | 1998-06-03 |
| EP0779815B1 (en) | 2001-03-28 |
| WO1996040187A1 (en) | 1996-12-19 |
| DE69612267T2 (en) | 2001-07-05 |
| ES2155188T3 (en) | 2001-05-01 |
| AU5802596A (en) | 1996-12-30 |
| EP0779815A1 (en) | 1997-06-25 |
| US5733538A (en) | 1998-03-31 |
| CA2197000A1 (en) | 1996-12-19 |
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