Polymer grafting by polysaccharide synthases

Abstract

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate synthase from Pasteurella multocida . The present invention also relates to coatings for biomaterials wherein the coatings provide protective properties to the biomaterial and/or act as a bioadhesive. Such coatings could be applied to electrical devices, sensors, catheters and any device which may be contemplated for use within a mammal. The present invention further relates to drug delivery matrices which are biocompatible and may comprise combinations of a biomaterial or a bioadhesive and a medicament or a medicament-containing liposome. The biomaterial and/or bioadhesive is a hyaluronic acid polymer produced by a hyaluronate synthase from Pasteurella multocida . The present invention also relates to the creation of chimeric molecules containing hyaluronic acid or hyaluronic acid—like chains attached to various compounds and especially carbohydrates or hydroxyl containing substances. The present invention also relates to a chondroitan synthase from Pasteurella multocida which is capable of producing polysaccharide polymers on an acceptor or primer molecule.

What I claim is:  
     
         1 . A method for transferring sugars to a primer molecule or an acceptor molecule utilizing an enzymatic process.  
     
     
         2 . The method according to  claim 1 , wherein the primer molecule or the acceptor molecule is a short polysaccharide.  
     
     
         3 . The method according to  claim 2 , wherein the polysaccharide is an oligosaccharide.  
     
     
         4 . The method according to  claim 1 , wherein the primer molecule or the acceptor molecule is a long polysaccharide.  
     
     
         5 . The method according to  claim 1 , wherein the primer molecule or the acceptor molecule is a solid substrate.  
     
     
         6 . The method according to  claim 5 , wherein the solid substrate is selected from the group consisting of a semiconductor, a metal, a natural or man-made polymer, and combinations thereof.  
     
     
         7 . The method according to  claim 1 , wherein the enzymatic process utilizes a polysaccharide synthase.  
     
     
         8 . The method according to  claim 1 , wherein the enzymatic process utilizes a glycosaminoglycan synthase.  
     
     
         9 . The method according to  claim 1 , wherein the enzymatic process utilizes a hyaluronic acid synthase.  
     
     
         10 . The method according to  claim 9 , wherein the hyaluronic acid synthase is selected from the group consisting of PmHAS-D, mutant variations of PmHAS, chemically modified versions of PmHAS, and combinations thereof.  
     
     
         11 . The method according to  claim 10 , wherein the amino acid sequence of the PmHAS-D is in accordance with SEQ ID NO:1.  
     
     
         12 . The method according to  claim 10 , wherein the nucleotide sequence of the PmHAS-D is in accordance with SEQ ID NO:2.  
     
     
         13 . The method of  claim 1 , wherein the enzymatic process utilizes a chondroitin synthase.  
     
     
         14 . The method of  claim 13 , wherein the chondroitin synthase is selected from the group consisting of PmCS, mutant variations of PmCS, chemically modified versions of PmCS, and combinations thereof.  
     
     
         15 . The method of  claim 14 , wherein the amino acid sequence of the PmCS is in accordance with SEQ ID NO: 3.  
     
     
         16 . The method of  claim 14 , wherein the nucleotide sequence of the PmCS is in accordance with SEQ ID NO: 4.  
     
     
         17 . The method of  claim 1 , wherein the sugars are selected from the group consisting of a natural or man-made monosaccharide, a GlcNAc, a GlcA, a GalNAc, a GalA, and combinations thereof.  
     
     
         18 . The method of  claim 1 , wherein the enzymatic process utilizes a polysaccharide synthase which has been altered by at least one condition selected from the group consisting of natural variations or variants, chemical modification, genetic modification, altered reaction conditions, and combinations thereof.  
     
     
         19 . A purified nucleic acid segment having a coding region encoding enzymatically active PmHAS-D.  
     
     
         20 . The purified nucleic acid segment of  claim 19 , wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:2.  
     
     
         21 . A purified nucleic acid segment having a coding region encoding enzymatically active PmHAS-D, wherein the purified nucleic acid segment is capable of. hybridizing to the nucleotide sequence of SEQ ID NO:2.  
     
     
         22 . A recombinant vector selected from the group consisting of a plasmid, a cosmid, a phage a virus vector, or a chromosomally integrated expression cassette and wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active PmHAS-D.  
     
     
         23 . The recombinant vector of  claim 22  wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:2.  
     
     
         24 . The recombinant vector of  claim 22 , wherein the recombinant vector is a plasmid.  
     
     
         25 . The recombinant vector of  claim 24 , wherein the plasmid further comprises an expression vector.  
     
     
         26 . The recombinant vector of  claim 25 , wherein the expression vector comprises a promoter operatively linked to the enzymatically active PmHAS-D coding region.  
     
     
         27 . A recombinant host cell, Wherein the recombinant host cell is a prokaryotic cell transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active PmHAS-D.  
     
     
         28 . A method for producing a polysaccharide polymer on a substrate, comprising the steps of: 
 providing a substrate having an immobilized polysaccharide primer thereon to provide a primed substrate;    combining the primed substrate with a PmHAS-D enzyme within a reaction medium, wherein the reaction medium contains at least one sugar precursor selected from the group consisting of UDP-GlcA and UDP-GlcNAc; and    reacting the PmHAS-D enzyme with the primed substrate to produce a substrate having a polysaccharide polymer coated thereon.    
     
     
         29 . The method of  claim 28 , wherein the polysaccharide polymer is hyaluronic acid.  
     
     
         30 . The method of  claim 28 , wherein the polysaccharide polymer is chondroitin.  
     
     
         31 . A substrate coated with a polysaccharide polymer manufactured according to the process of  claim 28 .  
     
     
         32 . The substrate of  claim 31 , wherein the polysaccharide polymer is hyaluronic acid.  
     
     
         33 . The substrate of  claim 31 , wherein the polysaccharide polymer is chondroitin.  
     
     
         34 . A method for producing a polysaccharide bioadhesive sealant, comprising the steps of: 
 combining an enzyme selected from the group consisting of PmHAS-D and PmCS with a reaction medium, wherein the reaction medium contains at least one sugar precursor selected from the group consisting of UDP-GlcA, UDP-GlcNAc or UDP-GalNAc; and    allowing the enzyme to react with the reaction medium, wherein the enzyme produces a polysaccharide polymer capable of use as a bioadhesive sealant by successively combining the at least one sugar precursor into a polymer chain.    
     
     
         35 . A polysaccharide bioadhesive sealant manufactured according to the process of  claim 34 .  
     
     
         36 . A method for producing a polysaccharide biomaterial containing a medicament delivery assembly, comprising the steps of: 
 preparing a polysaccharide polymer from an enzyme selected from the group consisting of PmHAS or PmCS, wherein the polysaccharide polymer is capable of acting as a bioadhesive;    providing at least one medicament delivery assembly containing one or more medicaments entrapped therein and deliverable within a wound site or a surgical site; and    mixing the prepared polysaccharide bioadhesive with the at least one medicament delivery assembly, wherein the prepared polysaccharide bioadhesive entraps the at least one medicament delivery. assembly to produce a polysaccharide biomaterial containing a medicament delivery system.    
     
     
         37 . A polysaccharide biomaterial containing a medicament delivery assembly manufactured according to the process of  claim 36 .  
     
     
         38 . A method for producing a polysaccharide biomaterial containing a medicament delivery system for administration at a wound, ulcer, injury or surgical site, comprising the steps of: 
 preparing a polysaccharide polymer from a enzyme selected from the group consisting of PmHAS or PmCS, wherein the prepared polysaccharide polymer is capable of acting as a bioadhesive;    providing at least one medicament delivery assembly containing one or more medicaments entrapped therein; and    mixing the prepared polysaccharide bioadhesive with the at least one medicament delivery assembly, wherein the prepared polysaccharide bioadhesive entraps the at least one medicament delivery assembly to provides a polysaccharide biomaterial containing a medicament delivery assembly.    
     
     
         39 . A polysaccharide biomaterial containing a medicament delivery system manufactured according to the process of  claim 38 .  
     
     
         40 . A method for inducing coagulation of a medicament delivery assembly-containing polysaccharide biomaterial composition at a site of injury, wound, or surgery in a mammal, thereby resulting in sealing and healing of the injury, wound or surgical site, comprising the steps of: 
 preparing a medicament delivery assembly-containing polysaccharide bioadhesive composition from an enzyme selected from the group consisting of PmHAS or PmCS; and    administering the medicament delivery assembly-containing polysaccharide bioadhesive composition to a mammal, wherein the coagulation of the medicament delivery assembly-containing polysaccharide bioadhesive composition forms a medium for entrapping the medicament delivery assembly at the site and embeds the medicament delivery assembly in the polysaccharide biomaterial composition.    
     
     
         41 . A method for hemostatically sealing and healing a wound or surgical incision and delivering to the wound or incision medicaments, comprising the steps of: 
 preparing a polysaccharide biomaterial composition having a medicament containing delivery assembly, wherein the polysaccharide bioadhesive composition is prepared from an enzyme selected from the group consisting of PmHAS and PmCS; and    administering the composition to the surface of a wound or incision, wherein coagulation of the composition forms a medium for entrapping the medicament delivery assembly at the site, embeds the medicament delivery assembly in the composition, and allows for a sustained release of the medicament delivery assembly contents to the wound or incision.    
     
     
         42 . A biomaterial composition, comprising: 
 an effective amount of a polysaccharide polymer produced from an enzyme selected from the group consisting of PmHAS-D and PmCS; and    at least one medicament delivery assembly containing at least one medicament, wherein the at least one medicament delivery assembly is embedded in the polysaccharide polymer so as to localize the at least one medicament within the polysaccharide polymer bioadhesive composition.