MAIMULT A TINKTUUS010000582B2 U U UTOTONI MIUI (12 ) United States Patent ( 10 ) Patent No. : US 10 ,000 ,582 B2 Jha et al. (45 ) Date of Patent: Jun . 19, 2018

(54 ) ETHYLSULFONATED HYALURONIC ACID (58 ) Field of Classification Search BIOPOLYMERS AND METHODS OF USE CPC ...... A61K 35 /30 ; A61K 35 /34 ; A61K 35 / 36 ; THEREOF A61K 35 / 545 ; A61K 9 / 4816 See application file for complete search history . (71 ) Applicant: The Regents of the University of California , Oakland , CA (US ) (56 ) References Cited U . S . PATENT DOCUMENTS ( 72 ) Inventors : Amit K . Jha , Oakland , CA (US ) ; Eda Isil Altiok , Berkeley, CA ( US ) ; Wesley 5 ,013 ,724 A 5 / 1991 Petitou et al. 5 ,652 ,014 A * 7 / 1997 Galin ...... A61L 27 /34 M . Jackson , Albany, CA (US ) ; Kevin 427 / 2 . 1 E . Healy ,Moraga , CA (US ) 6 ,051 , 701 A * 4 /2000 Cialdi ...... A61K 31/ 737 536 / 118 (73 ) Assignee: The Regents of the University of 6 , 288 ,043 B1* 9 / 2001 Spiro ...... A61L 27 / 20 California , Oakland , CA (US ) 424 /423 6 , 339, 074 B1 1 / 2002 Cialdi et al . 2009/ 0197797 AL 8 / 2009 Norbedo et al . @( * ) Notice : Subject to any disclaimer, the term of this 2010 /0278877 Al 11/ 2010 Tamura et al. patent is extended or adjusted under 35 2011/ 0046038 AL 2 /2011 Healy et al . U . S . C . 154 ( b ) by 189 days . FOREIGN PATENT DOCUMENTS @( 21 ) Appl. No. : 14 /780 ,447 WO WO 2009 / 120893 10 / 2009 (22 ) PCT Filed : Apr. 1, 2014 OTHER PUBLICATIONS PCT/ US2014 / 032528 ( 86 ) PCT No. : Abatangelo et al. ; “ Biocompatiblity and Enzymatic Degradation $ 371 (c )( 1 ), Studies on Sulphated Hyaluronic Acid Derivatives” ; Biomaterials ( 2 ) Date : Sep . 25 , 2015 18 : 1411, 1997 . Barbucci et al. , “ Sulfated Hyaluronic Acid as Heparin - like Material : (87 ) PCT Pub . No. : WO2014 / 165513 Physiochemical and Biological Characterization ” ; Journal of Mate rials Science : Materials in Medicine. 5 ( 11 ) : 830 - 833 , 1994 . PCT Pub . Date : Oct. 9 , 2014 Burdick , et al. ; “ Hyaluronic Acid Hydrogels for Biomedical Appli cations” ; Adv Mater . Mar. 25 , 2011 ;23 (12 ): H41 - 56 . (65 ) Prior Publication Data Cen et al. ; “ Assessment of in Vitro Bioactivity of Hyaluronic Acid and Sulfated Hyaluronic Acid Functionalized Electroactive Poly US 2016 /0053029 A1 Feb . 25 , 2016 mer” ; Biomacromolecules. 5 (6 ) : 2238 -2246 , 2004 . Necas et al. , “ Hyaluronic Acid ( Hyaluronan ): A Review ” ; Veterinarni Medicina , 53 ( 8 ) : 397 -411 , 2008 . Related U . S . Application Data Prestwich ; “ Hyaluronic Acid -Based Clinical Biomaterials Derived ( 60 ) Provisional application No . 61/ 807, 660 , filed on Apr. for Cell and Molecule Delivery in Regenerative Medicine” ; Journal 2 , 2013 . of Controlled Release . 155 (2 ): 193 - 199, 2011. (51 ) Int . CI. * cited by examiner C08B 37 /08 ( 2006 .01 ) Primary Examiner — Alma Pipic A6IK 35 /30 ( 2015 .01 ) (74 ) Attorney , Agent, or Firm — Bozicevic Field & A61K 35 / 34 ( 2015 .01 ) Francis , LLP ; Paula A . Borden A61K 35 /36 ( 2015 .01 ) A61K 35 /545 (2015 .01 ) (57 ) ABSTRACT C08L 5 /08 ( 2006 .01 ) The present disclosure provides methods for sulfonation of A61K 9 / 48 ( 2006 . 01) hyaluronic acid . The present disclosure provides sulfonated (52 ) U . S . CI. hyaluronic acid , and compositions , including pharmaceuti CPC ...... C08B 37/ 0072 (2013 . 01 ) ; A61K 9 /4816 cal compositions , comprising the sulfonated hyaluronic ( 2013 . 01 ) ; A61K 35 /30 (2013 . 01 ) ; A61K 35 / 34 acid . The present disclosure provides implantable materials ( 2013 .01 ); A61K 35 / 36 (2013 . 01 ); A61K and drug delivery compositions comprising a subject sul 35 /545 (2013 . 01 ) ; CO8L 5/ 08 ( 2013 .01 ) ; C12N fonated hyaluronic acid . 2533/ 80 ( 2013 .01 ) 17 Claims, 15 Drawing Sheets U. S . Patentatent Jun . 19 , 2018 Sheet 1 of 15 US 10 , 000 ,582 B2

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Nuclei /CD31 US 10 , 000 , 582 B2 ETHYLSULFONATED HYALURONIC ACID fonated hyaluronic acid , and compositions, including phar BIOPOLYMERS AND METHODS OF USE maceutical compositions , comprising the sulfonated THEREOF hyaluronic acid . The present disclosure provides implant able materials and drug delivery compositions comprising a CROSS -REFERENCE 5 subject sulfonated hyaluronic acid . This application claims the benefit of U .S . Provisional BRIEF DESCRIPTION OF THE DRAWINGS Patent Application No . 61 / 807 ,660 , filed Apr. 2 , 2013 ,which application is incorporated herein by reference in its entirety . FIG . 1A depicts HyA and ES -HyA degradation . 10 FIG . 1B depicts average molecular weight of unsulfated INTRODUCTION versus sulfated HyA following hyaluronidase treatment . FIG . 2 depicts combustion analysis of unsulfated HyA Hyaluronic acid (HyA ) is a natural glycosaminoglycan and sulfated HyA . (GAG ) biopolymer with a variety of favorable biological FIG . 3 depicts efficiency of EMCH conjugation to unsul properties , such as promoting cell growth , inhibiting throm - 15 fated HyA and sulfated HyA ( ES -HyA ) . bosis and modulating the tissue distribution of secreted FIGS . 4A - B depict the synthesis of sulfated hyaluronic growth factors . It is currently used in a variety of products acid . to improve surface biocompatibility , as a substrate for tissue FIGS . 5A - B depict Proton ( H ) NMR validation of engineering and to control drug pharmacodynamics . A sig - hyaluronic acid sulfation . nificant limiting factor for the use of HyA in medical 20 FIG . 6 depicts Proton ( ' H ) - Carbon (13C ) NMR validation applications is its degradation in vivo due to the high of hyaluronic acid sulfation . endogenous concentration of hyaluronidase and other gly - FIG . 7 depicts functional validation of hyaluronic acid colytic proteins. Depending on the tissue, the half - life of sulfation by platelet adhesion assay . HyA can be as short as minutes , and when in contact with FIG . 8 depicts functional validation of hyaluronic acid the blood, its half - life is approximately 1 . 5 hours. Therefore, 25 sulfation by platelet poor plasma clotting assay . current uses of HyA are limited to applications where its FIG . 9 depicts functional validation of hyaluronic acid biomaterial properties are only required for a short duration sulfation by carbazole assay . of time. FIG . 10 depicts functional validation of hyaluronic acid Sulfation is a natural means of preserving GAG biopo - sulfation by retention kinetics assay. lymers. In vivo , enzymes replace the native hydroxyl groups 30 FIG . 11 depicts the effects of sulfation of hyaluronic acid on the GAG disaccharide unit with sulfates , and as a result, on cell viability , proliferation and adhesion . the biopolymer resists the activity of glycolytic proteins . FIG . 12 depicts the effects of sulfation of hyaluronic acid Two commonly sulfated GAGs include heparin sulfate and on cell differentiation . chondroitin sulfate . Both of these compounds are used in medical applications to take advantage of their enhanced 35 DEFINITIONS stability . However, typical high - end molecular weights for heparin sulfate and chondroitin sulfate are typically in the The terms “ subject ,” “ individual, " " host ,” and “ patient” range of 30 kDa and 150 kDa , respectively . The potential are used interchangeably herein to a member or members of uses for the sulfated GAG biopolymers are therefore limited any mammalian or non - mammalian species. Subjects and in comparison to HyA , which is routinely synthesized with 40 patients thus include , without limitation , humans , non - hu molecular weights exceeding 1 . 0 MDa . However , Hya is the man primates , canines , felines , ungulates ( e . g ., equine , only GAG that does not become sulfated naturally . bovine , swine ( e . g ., pig ) ) , avians, rodents ( e . g . , rats , mice ) , To improve the stability of HyA in vivo there are several and other subjects . Non -human animal models , particularly methods of synthetic GAG sulfation . However, the primary mammals , e . g . a non -human primate , a murine ( e . g . , a drawback to current sulfation methods is that the chemical 45 mouse , a rat ) , lagomorpha , etc . may be used for experimen reactions must be performed in organic solvents that sig tal investigations. nificantly complicate the process of translating the final “ Treating ” or “ treatment” of a condition or disease product into a biocompatible formulation . Furthermore , a includes : ( 1 ) preventing at least one symptom of the condi high degree of sulfation ( substitution ratios of 2 - 4 sulfates tion , i. e . , causing a clinical symptom to not significantly per disaccharide unit ) is required to satisfactorily resist 50 develop in a mammal that may be exposed to or predisposed enzymatic degradation . Thus , extensive modification of the to the disease but does not yet experience or display symp HyA molecule limits the ability to perform additional chemi- toms of the disease , ( 2 ) inhibiting the disease , i . e . , arresting cal reactions on the biopolymer to improve its utility . or reducing the development of the disease or its symptoms, There is a need in the art for methods of increasing the in or ( 3 ) relieving the disease , i. e ., causing regression of the vivo half- life of hyaluronic acid . 55 disease or its clinical symptoms. A “ therapeutically effective amount” or “ efficacious LITERATURE amount" means the amount of a compound that, when administered to a mammal or other subject for treating a U . S . Pat. No. 6 , 338 ,074 ; U . S . Pat . No. 5 , 013 ,724 ; Aba disease , is sufficient, in combination with another agent , or tangelo et al. ( 1997 ) Biomaterials 18 : 1411 ; Barbucci et al. 60 alone in one or more doses , to effect such treatment for the ( 1994 ) Journal ofMaterials Science : Materials in Medicine . disease . The “ therapeutically effective amount ” will vary 5: 830 ; Necas et al (2008 ) Veterinarni Medicina , 53 (8 ) : 397 depending on the compound, the disease and its severity and the age, weight, etc ., of the subject to be treated . SUMMARY The term “ unit dosage form , " as used herein , refers to 65 physically discrete units suitable as unitary dosages for The present disclosure provides methods for sulfonation human and animal subjects , each unit containing a prede of hyaluronic acid . The present disclosure provides sul- termined quantity of compounds of the present invention US 10 ,000 , 582 B2 calculated in an amount sufficient to produce the desired within the invention . The upper and lower limits of these effect in association with a pharmaceutically acceptable smaller ranges may independently be included in the smaller diluent, carrier or vehicle . The specifications for the novel ranges , and are also encompassed within the invention , unit dosage forms of the present invention depend on the subject to any specifically excluded limit in the stated range . particular compound employed and the effect to be achieved , 5 Where the stated range includes one or both of the limits , and the pharmacodynamics associated with each compound ranges excluding either or both of those included limits are in the host. also included in the invention . The term “ physiological conditions ” is meant to encom pass those conditions compatible with living cells , e . g . , Unless defined otherwise , all technical and scientific predominantly aqueous conditions of a temperature, pH , 10 terms used herein have the same meaning as commonly salinity, etc. that are compatible with living cells . understood by one of ordinary skill in the art to which this A “ pharmaceutically acceptable excipient, " " pharmaceu invention belongs. Although any methods and materials tically acceptable diluent, " " pharmaceutically acceptable similar or equivalent to those described herein can also be carrier, ” and “ pharmaceutically acceptable adjuvant” means used in the practice or testing of the present invention , the an excipient, diluent, carrier , and adjuvant that are useful in 15 preferred methods and materials are now described . All preparing a pharmaceutical composition that are generally publications mentioned herein are incorporated herein by safe , non - toxic and neither biologically nor otherwise unde - reference to disclose and describe the methods and / or mate sirable , and include an excipient, diluent, carrier, and adju - rials in connection with which the publications are cited . vant that are acceptable for veterinary use as well as human It must be noted that as used herein and in the appended pharmaceutical use. “ A pharmaceutically acceptable excipi- 20 claims, the singular forms “ a ,” “ an ,” and “ the ” include plural ent, diluent, carrier and adjuvant” as used in the specification referents unless the context clearly dictates otherwise . Thus , and claims includes one and more than one such excipient, for example , reference to " a sulfonated hyaluronic acid diluent, carrier, and adjuvant. matrix ” includes a plurality of such matrices and reference As used herein , the term " label moiety ” is intended to to " the composition ” includes reference to one or more mean one or more atoms that can be specifically detected to 25 compositions and equivalents thereof known to those skilled indicate the presence of a substance to which the one or in the art , and so forth . It is further noted that the claimsmay more atom is attached . A label moiety can be a primary label be drafted to exclude any optional element. As such , this that is directly detectable or secondary label that can be statement is intended to serve as antecedent basis for use of indirectly detected , for example , via interaction with a such exclusive terminology as " solely , " " only ” and the like primary label . Exemplary primary labels include , without 30 in connection with the recitation of claim elements , or use of limitation , an isotopic label such as a naturally non - abundant a “ negative” limitation . heavy isotope or radioactive isotope , examples of which It is appreciated that certain features of the invention , include 14C , 1231, 124119 , 1251, 1311 , 32P , 35S or 3H ; optically which are , for clarity , described in the context of separate detectable moieties such as a chromophore, luminophore , embodiments , may also be provided in combination in a fluorophore , quantum dot or nanoparticle ; electromagnetic 35 single embodiment. Conversely , various features of the spin label; calorimetric agent; magnetic substance ; electron - invention , which are , for brevity , described in the context of rich material such as a metal; electrochemiluminescent label a single embodiment, may also be provided separately or in such as Ru (bpy ) , 2 + ; moiety that can be detected based on a any suitable sub - combination . All combinations of the nuclear magnetic , paramagnetic , electrical, charge to mass , embodiments pertaining to the invention are specifically or thermal characteristic ; or light scattering or plasmon 40 embraced by the present invention and are disclosed herein resonant materials such as gold or silver particles . Fluoro - just as if each and every combination was individually and phores that are useful in the invention include , for example, explicitly disclosed . In addition , all sub -combinations of the fluorescent lanthanide complexes, including those of Euro - various embodiments and elements thereof are also specifi pium and Terbium , fluorescein , fluorescein isothiocyanate , cally embraced by the present invention and are disclosed carboxyfluorescein (FAM ), dichlorotriazinylamine fluores - 45 herein just as if each and every such sub - combination was cein , rhodamine , tetramethylrhodamine , umbelliferone , individually and explicitly disclosed herein . eosin , erythrosin , coumarin , methyl- coumarins, pyrene , The publications discussed herein are provided solely for Malacite green , Cy3 , Cy5 , stilbene , Lucifer Yellow , Cascade their disclosure prior to the filing date of the present appli BlueTM Texas Red , alexa dyes , dansyl chloride , phyco - cation . Nothing herein is to be construed as an admission erythin , green fluorescent protein and its wavelength shifted 50 that the present invention is not entitled to antedate such variants , bodipy, and others known in the art such as those publication by virtue of prior invention . Further, the dates of described in Haugland , Molecular Probes Handbook , (Eu - publication provided may be different from the actual pub gene , Oreg . ) 6th Edition ; The Synthegen catalog (Houston , lication dates which may need to be independently con Tex . ), Lakowicz , Principles of Fluorescence Spectroscopy, firmed . 2nd Ed . , Plenum Press New York ( 1999 ) , or WO 98 / 59066 . 55 Before the present invention is further described , it is to DETAILED DESCRIPTION be understood that this invention is not limited to particular embodiments described , as such may, of course , vary. It is The present disclosure provides methods for sulfonation also to be understood that the terminology used herein is for of hyaluronic acid . The present disclosure provides sul the purpose of describing particular embodiments only , and 60 fonated hyaluronic acid , and compositions , including phar is not intended to be limiting, since the scope of the present maceutical compositions, comprising the sulfonated invention will be limited only by the appended claims. hyaluronic acid . The present disclosure provides implant Where a range of values is provided , it is understood that able materials and drug delivery compositions comprising a each intervening value , to the tenth of the unit of the lower subject sulfonated hyaluronic acid . limit unless the context clearly dictates otherwise, between 65 Methods for Sulfonation of Hyaluronic Acid the upper and lower limit of that range and any other stated in certain embodiments , a method for sulfonation of or intervening value in that stated range , is encompassed hyaluronic acid is provided . Hyaluronic acid is a polymer of US 10 ,000 ,582 B2 disaccharides, themselves composed of D - glucuronic acid sodium periodate oxidation . In certain embodiments sodium and D - N -acetylglucosamine , linked via alternating B - 1 , 4 periodate oxidation of vicinal diols is achieved by introduc and B - 1 , 3 glycosidic bonds . ing sodium periodate to a reaction mixture that contains In certain embodiments , the method includes activating vicinal diols . Sodium periodate oxidation be accomplished one or more carbonyl groups in a hyaluronic acid polymer. 5 using a variety of reaction conditions. For example , in some Activating the carbonyl group may produce an activated embodiments , sodium periodate oxidation is performed at carbonyl group in the hyaluronic acid polymer. room temperature and the reaction is protected from light , Activating the carbonyl group in the hyaluronic acid e . g , performed in the dark . In some instances, reaction polymer may be accomplished using a variety of methods. conditions are modified in order to increase or reduce the For example , in some embodiments , the activating includes 10 production of reactive carbonyl groups produced by a par coupling a coupling agent to the carbonyl group to produce ticular sodium periodate oxidation reaction . Those of skill in a coupling agent- modified hyaluronic acid polymer . As such , the art will understand that reaction conditions for sodium the activated carbonyl group may include a carbonyl group periodate oxidation may be modified in order to optimize of the hyaluronic acid polymer attached ( e. g ., bonded , such reaction output, reaction convenience , or any other reaction as covalently bonded ) to the coupling agent . The coupling 15 parameter of interest. agent can be any of a variety of coupling agents that provide The reactive carbonyl groups ( e . g . , reactive aldehyde for attachment of the coupling agent to the carbonyl group groups ) may then be coupled to the sulfonate - containing of the hyaluronic acid polymer. For instances , the coupling moiety to produce the sulfonated hyaluronic acid polymer . agentmay provide for attachment to a carbonyl group , such As such , the method may include the step where the attach as a carboxyl group or an aldehyde group . In certain 20 ing includes coupling the sulfonate -containing moiety to the instances, the coupling agent is configured for attachment to reactive carbonyl group to produce the sulfonated a carboxyl group in the hyaluronic acid polymer . In these hyaluronic acid polymer . In these embodiments , the sul instances , the coupling agent may include a moiety that is fonate - containing moiety may be coupled directly to the reactive towards a carbonyl group , such as a carboxyl- hyaluronic acid polymer without an intervening coupling reactive moiety . For example , the coupling agent may 25 group . include adipic acid dihydrazide . Coupling of the sulfonate -containing moiety to the reac In certain embodiments, the method further includes tive carbonyl group to produce the sulfonated hyaluronic attaching a sulfonate - containing moiety to the activated acid polymer may be accomplished using a variety of carbonyl group to produce a sulfonated hyaluronic acid methods . For example , in some embodiments an aldehyde polymer . As discussed above , coupling the coupling group to 30 derivative of hyaluronic acid is first produced by methods a carbonyl group of the hyaluronic acid polymer may described herein and reactive carbonyl groups of the alde produce a coupling agent -modified hyaluronic acid polymer. hyde derivative of hyaluronic acid are coupled to sulfonate In these embodiments, attaching the sulfonate - containing containing moieties . In some embodiments , the coupled moiety to the activated carbonyl group includes attaching molecule is further reduced after coupling by a reducing the sulfonate - containing moiety to the coupling agent- modi - 35 agent . Any convenient reducing agent useful in reducing the fied hyaluronic acid polymer to produce the sulfonated coupled molecule to produce a sulfonated hyaluronic acid hyaluronic acid polymer. As such , the sulfonate -containing polymer may be used . For example , in some embodiments moiety is indirectly coupled to the carbonyl group of the the reducing agent may be a mild reducing agent, e . g . , hyaluronic acid polymer through the coupling agent. For sodium cynoborohydride . instance , one end of the coupling agent may be attached to 40 In certain embodiments , the method is performed in an the hyaluronic acid polymer , as described above in the first aqueous reaction mixture . By “ aqueous ” is meant a solution step of the presently disclosed method . The other end of the or mixture where the primary solvent is water. In certain coupling agent may be attached to the sulfonate - containing instances , an aqueous reaction mixture may facilitate the moiety , as described in the second step of the presently production of a sulfonated hyaluronic acid polymer in a disclosed method . 45 pharmaceutically acceptable solution . In certain embodi In other embodiments , the first step of the method , ments , the aqueous reaction mixture has a pharmaceutically activating the carbonyl group in the hyaluronic acid poly - acceptable pH . For example, the reaction mixture may have mer , includes producing at least one reactive carbonyl group a pH of 5 to 7 . on the hyaluronic acid polymer. For instance , the reactive in certain embodiments, the method finds use in the carbonyl group may include a reactive aldehyde group . In 50 production of a sulfonated hyaluronic acid polymer with a some instances , the reactive carbonyl group may be pro - high molecular weight. For example , the hyaluronic acid duced on one or both of the saccharides that make up the polymer may have a molecular weight of 100 kDa or more , disaccharide units of hyaluronic acid . For example , the such as 200 kDa or more, including 300 kDa or more, or 400 reactive carbonyl groups may be produced on one or more kDa or more , or 500 kDa or more , or 600 kDa or more , or of the D - glucuronic acid subunits of hyaluronic acid , or may 55 700 kDa or more , or 800 kDa or more , or 900 kDa or more , be produced on one or more of the D - N - acetylglucosamine or 1 MDa or more , or 1 . 5 MDa or more . In some instances , subunits of hyaluronic acid . In certain embodiments , the the hyaluronic acid has a molecular weight of 400 kDa or reactive carbonyl groups are produced on the D - glucuronic more . In some instances, the hyaluronic acid polymer has a acid subunits of hyaluronic acid . In these instances , the molecular weight of 1 MDa or more . reactive carbonyl groups ( e. g ., reactive aldehyde groups) 60 In certain embodiments , the method is performed at a may be produced by cleavage of the D - glucuronic acid ring . temperature ranging from 10° C . to 40° C ., such as from 15° For example , vicinal diols of the D - glucuronic acid ring may C . to 35º C . , or from 15° C ., to 30° C ., or from 15° C . to 25° be cleaved to form two aldehyde groups from the D - glu - C . In certain embodiments , the method is performed at room curonic acid subunit of hyaluronic acid . temperature , e . g ., standard room temperature , such as about Cleavage of vicinal diols may be may be accomplished 65 20° C . using a variety of methods . For example , in some embodi- In certain embodiments , the sulfonated hyaluronic acid ments , the methods of cleaving vicinal diols includes polymer has a particular sulfonate :disaccharide ratio . By US 10 , 000 , 582 B2 " sulfonate : disaccharide ratio is meant the average number of limited to , crosslinkers, proteins, peptides , labels ( e . g ., dyes , sulfonate groups per disaccharide units of the sulfonated fluorescent labels , etc .) , drugs , and the like. hyaluronic acid polymer. For example , a sulfonate :disaccha - Sulfonated Hyaluronic Acid ride ratio of 1 indicates that there are , on average , one Embodiments of the present disclosure further include a sulfonate groups per disaccharide units in the sulfonated 5 sulfonated hyaluronic acid polymer produced by the meth hvaluronic acid polymer. By “ average ” is meant the arith - ods described herein . Embodiments of the present disclosure include a sulfonated hyaluronic acid matrix ( a “ sulfonated metic mean . In certain instances , the sulfonated hyaluronic HyA matrix ” ) . A subject sulfonated HyA matrix comprises acid polymer has a sulfonate :disaccharide ratio of 2 or less , a sulfonated HyA polymer, as discussed above , and has the such as 1 . 7 or less, or 1 . 5 or less , or 1 .3 or less , or 1 or less , 10 properties discussed above. such as 0 .9 or less , or 0 .8 or less, or 0 .7 or less , or 0 .6 or less, In certain instances , the sulfonated hyaluronic acid poly or 0 .5 or less, or 0 .4 or less, or 0 . 3 or less, or 0 .2 or less, or mer has a sulfonate :disaccharide ratio of 2 or less , such as 0 . 1 or less . In some instances , the sulfonated hyaluronic acid 1 . 7 or less , or 1 . 5 or less , or 1 . 3 or less, or 1 or less, such as polymer has a sulfonate :disaccharide ratio of 1 or less . In 0 . 9 or less , or 0 . 8 or less , or 0 . 7 or less, or 0 .6 or less , or 0 . 5 some instances , the sulfonated hyaluronicronic acid polymerpolymer has 15 or less , or 0 .4 or less, or 0 .3 or less, or 0 .2 or less , or 0 .1 or a sulfonate : disaccharide ratio of 0 . 5 or less . In some less. In some instances , the sulfonated hyaluronic acid instances, the sulfonated hyaluronic acid polymer has a polymer has a sulfonate : disaccharide ratio of 1 or less . In sulfonate : disaccharide ratio of 0 . 1 or less . some instances, the sulfonated hyaluronic acid polymer has In certain embodiments , the sulfonate - containing moiety a sulfonate : disaccharide ratio of 0 . 5 or less. In some includes a reactive group and a sulfonate group . The reactive 20 instances , the sulfonated hyaluronic acid polymer has a group may be any convenient reactive group that provides sulfonate : disaccharide ratio of 0 . 1 or less. from reaction ( e . g ., coupling reaction ) between the sul In certain embodiments , the sulfonated hyaluronic acid fonate -containing moiety and the coupling agent (e . g ., the polymer has a degradation half - life significantly longer that coupling agent coupled to the hyaluronic acid polymer ) or an un - sulfonated hyaluronic acid polymer . By degradation the reactive carbonyl group of the hyaluronic acid polymer 25 half - life is meant the time in which it takes half of the as described above . For example , as described above , the sulfonated hyaluronic acid polymer to degrade . For coupling agent may include a hydrazide reactive group . In example , the sulfonated hyaluronic acid polymer may have these instances , the sulfonate - containing moiety may a half- life of 5 hours or more , such as 6 hours or more , or include a reactive group ( e . g . , a reactive group that reacts 7 hours or more , or 8 hours or more , or 9 hours or more , or with a hydrazide group ), such as an aldehyde group . For 30 10 hours or more , or 12 hours or more , or 15 hours or more , instance , the sulfonate - containing moiety may include or 17 hours or more , or 20 hours or more , or 22 hours or 3 - oxopropane - 1 - sulfonate . In other embodiments , as more , or 24 hours or more , or 25 hours or more , or 27 hours described above , the method may include producing a or more , or 30 hours or more , or 35 hours or more , or 40 reactive carbonyl group on the hyaluronic acid polymer, hours or more , or 45 hours or more , or 48 hours or more . In such as a reactive aldehyde group . In these embodiments , 35 some instances , the sulfonated hyaluronic acid polymer has the reactive group of the sulfonate - containing moiety may an in vivo half - life of 10 hours or more . be configured to react with the reactive carbonyl group ( e . g . , subject sulfonated hyaluronic acid polymer can further reactive aldehyde group ) of the hyaluronic acid polymer. For include one or more additional moieties , e . g ., crosslinkers , instance , the sulfonate -containing moiety may include an proteins, peptides , labels ( e. g ., dyes, fluorescent labels , etc . ), amino group . In these instances , examples of the sulfonate - 40 drugs , and the like . containing moiety may include 2 - aminoethanesulfonate or Embodiments of the present disclosure further include a 2 - aminoethyl hydrogen sulfate . pharmaceutical composition that includes a sulfonated In certain embodiments , the sulfonated hyaluronic acid hyaluronic acid polymer as described herein . The pharma polymer has a degradation half - life significantly longer that ceutical compositions may also include a pharmaceutically an un - sulfonated hyaluronic acid polymer . By degradation 45 acceptable buffer . The pharmaceutical compositions may half- life is meant the time in which it takes half of the also include one or more of an excipient, solubilizer, stabi sulfonated hyaluronic acid polymer to degrade . For lizer , buffer , tonicity modifier , bulking agent, viscosity example , the sulfonated hyaluronic acid polymer may have enhancer/ reducer , surfactant, chelating agent, adjuvant, an in vivo half- life of 5 hours or more, such as 6 hours or combinations thereof, and the like . more , or 7 hours or more , or 8 hours or more , or 9 hours or 50 As noted above , a subject sulfonated hyaluronic acid more , or 10 hours or more , or 12 hours or more , or 15 hours polymer can further include one or more additionalmoieties , or more , or 17 hours or more , or 20 hours or more , or 22 e .g . , crosslinkers , polypeptides, labels ( e .g . , dyes , fluores hours or more , or 24 hours or more , or 25 hours or more , or cent labels, etc . ) , drugs, and the like . Such moieties can be 27 hours or more , or 30 hours or more , or 35 hours or more , conjugated to the sulfonated hyaluronic acid polymer, to or 40 hours or more , or 45 hours or more , or 48 hours or 55 form a sulfonated hyaluronic acid polymer conjugate . more . In some instances , the sulfonated hyaluronic acid Polypeptides that are of interest for attachment to a polymer has an in vivo half - life of 10 hours or more . sulfonated hyaluronic acid polymer , to generate a subject In certain embodiments , the method further includes polypeptide - polymer conjugate include , e . g . , growth factors , conjugating the sulfonated hyaluronic acid polymer to one receptors, polypeptide ligands for receptors , enzymes , anti or more moieties of interest . For example , embodiments of 60 bodies , coagulation factors , anti - coagulation factors, angio the sulfonated hyaluronic acid may have a low sulfonate : genic factors , anti - angiogenic factors , etc . Suitable polypep disaccharide ratio as described above . In these embodi- tides include linear polypeptides and cyclic polypeptides . ments , there may be a plurality of unmodified disaccharide Suitable polypeptides include naturally occurring polypep units in the hyaluronic acid polymer . In some instances , tides, synthetic polypeptides, and the like . Polypeptides that these unmodified disaccharide units may be able to partici - 65 are of interest for attachment to a sulfonated hyaluronic acid pate in conjugation reactions with other functionalized moi- polymer , to generate a subject polypeptide- polymer conju eties of interest. Moieties of interest include , but are not gate include polypeptides having a molecular weight of from US 10 ,000 ,582 B2 10 about 1 kDa to about 2000 kDa , e . g ., from about 1 kDa to vasoactive intestinal peptide, angiogenin , angiotropin , about 2 kDa, from about 2 kDa to about 2 . 5 kDa, from about fibrin ; hirudin ; a leukemia inhibitory factor ; an IL - 1 receptor 2 . 5 kDa to about 5 kDa, from about 5 kDa to about 10 kDa , antagonist ( e . g ., Kineret® (anakinra )) ; an ion channel , e .g . , from about 10 kDa to about 25 kDa, from about 25 kDa to cystic fibrosis transmembrane conductance regulator about 50 kDa, from about 50 kDa to about 100 kDa, from 5 (CFTR ); dystrophin ; utrophin , a tumor suppressor; lyso about 100 kDa to about 250 kDa, from about 250 kDa to somal enzyme acid a - glucosidase (GAA ) ; and the like . about 500 kDa, from about 500 kDa to about 1000 kDa, Suitable polypeptides include sonic hedgehog (Shh ) , bone from about 1000 kDa to about 2000 kDa . In some cases , the morphogenic protein - 4 , interleukin -3 ( IL - 3 ), stem cell fac polypeptide has a molecular weight greater than 2000 kDa tor - 1 (SCF - 1 ) , fms- like tyrosine kinase - 3 (Fit3 ) ligand , Suitable polypeptides include , but are not limited to , an 10 leukemia inhibitory factor (LIF ) , fibroblast growth factor - 2 interferon ( e . g ., IFN - Y , IFN - a , IFN - B , IFN - W ; IFN - T ) ; an (FGF - 2 ) , and epidermal growth factor ( EGF ) . Suitable poly insulin ( e . g . , Novolin , Humulin , Humalog , Lantus, Ultra - peptides include brain - derived neurotrophic factor (BDNF ) , lente , etc . ) ; an erythropoietin ( “ EPO ” ; e . g . , Procrit® , nerve growth factor (NGF ) , neurotrophin - 3 (NT - 3 ) , neuro Eprex® , or Epogen® ( epoetin - a ) ; Aranesp® (darbepoietin - trophin -4 (NT - 4 ) , neurotrophin - 5 (NT - 5 ) , basic fibroblast a ); Neo Recormon® , Epogin® ( epoetin - B ) and the like ) ; an 15 growth factor (bFGF ) , insulin -like growth factor- 1 ( IGF - 1 ) , antibody ( e . g . , a monoclonal antibody ) ( e . g ., Rituxan® glial- derived neurotrophic factor GDNF( ) , and protease ( rituximab ); Remicade® (infliximab ) ; Herceptin® ( trastu - nexin - 1 . Suitable angiogenic polypeptides include a netrin - 1 zumab ); HumiraTM (adalimumab ); Xolair® (omalizumab ); polypeptide , a vascular endothelial growth factor (VEGF ) Bexxar® ( tositumomab ); RaptivaTM (efalizumab ); polypeptide, a platelet- derived growth factor (PDGF ) poly ErbituxTM (cetuximab ) ; and the like ), including an antigen - 20 peptide , a fibroblast growth factor (FGF ) polypeptide , and binding fragment of a monoclonal antibody ; a blood factor ( e . g ., Activase® ( alteplase) tissue plasminogen activator; Suitable polypeptides also include clotting factors , e . g ., NovoSeven® ( recombinant human factor VIIa ); Factor thrombin , etc . Suitable polypeptides also include anti - co VIIa ; Factor VIII ( e . g . , Kogenate? ) ; Factor IX ; B -globin ; agulants. Suitable polypeptides also include cell -binding hemoglobin , and the like ) ; a colony stimulating factor ( e . g ., 25 polypeptides . Neupogen® ( filgrastim ; G -CSF ) ; Neulasta (pegfilgrastim ); Suitable polypeptides also include, e. g. ,Nestin , Vimentin , granulocyte colony stimulating factor ( G - CSF ) , granulo Prominin / CD133 , Sonic hedgehog and other hedgehog cyte -monocyte colony stimulating factor, macrophage ligands , Wnt ligands, Neurocan /tenascin C , Nurr 1 , Pax - 6 , colony stimulating factor, megakaryocyte colony stimulat Sox - 2 , Musashi - 1 , NG2/ CSPG - 4 , Neuro D3 , Neurogenin 1 , ing factor; and the like ) ; a growth hormone ( e . g . , a soma - 30 and active fragments and subsequences of any these poly totropin , e . g . , Genotropin® , Nutropin® , Norditropin® , peptides . Saizen® , Serostim® , Humatrope® , etc . , a human growth Suitable polypeptides also include, e . g . , B tubulin III , hormone; and the like ); an interleukin ( e. g ., IL - 1 ; IL - 2 , MAP2 , Neuron specific enolase, NCAM , CD24 , HAS , Syn including , e . g . , Proleukin® ; IL - 3 , IL - 4 , IL - 5 , IL - 6 , IL - 7 , apsin I, Synaptophysin , CAMK Iia , Tyrosine hydroxylase , IL - 8 , IL - 9 ; etc . ); a growth factor ( e . g ., Regranex® (becla - 35 Glutamate transporter, Glutamate receptor , Choline rececp permin ; PDGF ) ; Fiblast ( trafermin ; bFGF ) ; Stemgen® tor, nicotinic A2, EphB2, GABA - A receptor, Serotonin ( ancestim ; stem cell factor ) ; keratinocyte growth factor; an ( 5HT- 3 ) receptor, Choline acetyltransferase , and fragments acidic fibroblast growth factor, a stem cell factor, a basic and subsequences of any of the foregoing . fibroblast growth factor, a hepatocyte growth factor; and the Suitable polypeptides also include, e . g . , a calcium chan like ) ; a receptor ( e . g ., a TNF - a - binding soluble receptor 40 nel ; a T - cell antigen receptor; a chemokine receptor ; a such as Enbrel® ( etanercept) ; a VEGF receptor; a interleu - potassium channel ; a neurotransmitter receptor ( e . g ., a sero kin receptor ; a y / d T cell receptor; and the like ); a neu - tonin receptor ; a GABA receptor; a glutamate receptor, a rotransmitter receptor ( e . g . , a nicotinic acetylcholine recep - nicotinic acetylcholine receptor , etc . ) ; a growth factor recep tor, a glutamate receptor, a GABA receptor, etc .) ; an enzyme tor (e . g ., epidermal growth factor receptor; vascular ( e . g . , a - glucosidase ; Cerazyme ( imiglucarase ; B - glucoce - 45 endothelial growth factor receptor, etc . ) ; a bone morphoge rebrosidase , Ceredase® ( alglucerase ); an enzyme activator netic protein ; a polypeptide that activates a cell signaling ( e . g . , tissue plasminogen activator ) ; a chemokine ( e . g . , pathway ; an antibody ; and the like . IP - 10 ; Mig ; Groa / IL - 8 , RANTES ;MIP - la ; MIP - 1B ;MCP - Suitable drugs include, but are not limited to , cytotoxic 1 ; PF - 4 ; and the like) ; an angiogenic agent ( e . g . , vascular compounds ( e . g . , cancer chemotherapeutic compounds ) ; endothelial growth factor (VEGF ) ; an anti - angiogenic agent 50 antiviral compounds; biological response modifiers ( e . g . , ( e . g . , a VEGF receptor ); a neuroactive peptide such as hormones , chemokines, cytokines , interleukins , etc . ) ; micro bradykinin , cholecystokinin , gastin , secretin , oxytocin , tubule affecting agents ; hormone modulators ; steroidal com gonadotropin -releasing hormone , beta - endorphin , enkeph pounds ; and the like . Suitable drugs include those listed alin , substance P , somatostatin , prolactin , galanin , growth hereinbelow ( e . g . , lipid - regulating agents ; sex hormones; hormone - releasing hormone , bombesin , dynorphin , neuro - 55 androgenic agents ; antihypertensive agents ; anti- diabetic tensin ,motilin , thyrotropin , neuropeptide Y , luteinizing hor agents ; anti- viral agents , as described below ). Suitable drugs monem , calcitonin , insulin , glucagon , vasopressin , angio - include cancer chemotherapeutic agents . tensin II, thyrotropin - releasing hormone , vasoactive Suitable antibodies ( e . g . , for use in cancer treatment ) intestinal peptide , a sleep peptide , etc . ; other proteins such include, but are not limited to , naked antibodies , e . g . , as a thrombolytic agent, an atrial natriuretic peptide , bone 60 trastuzumab (Herceptin ) , bevacizumab ( AvastinTM ) , cetux morphogenic protein , thrombopoietin , relaxin , glial fibril - imab (ErbituxTM ) panitumumab (VectibixTM ) , Ipilimumab lary acidic protein , follicle stimulating hormone , a human ( YervoyTM ) , rituximab ( Rituxan ) , alemtuzumab alpha - 1 antitrypsin , a leukemia inhibitory factor, a trans (LemtradaTM ) , Ofatumumab ( ArzerraTM ), Oregovomab forming growth factor, an insulin - like growth factor, a (OvaRexTM ) , Lambrolizumab (MK - 3475 ) , pertuzumab (Per luteinizing hormone , a macrophage activating factor, tumor 65 jetaTM ), ranibizumab (LucentisTM ) etc ., and conjugated anti necrosis factor, a neutrophil chemotactic factor, a nerve bodies , e . g . , gemtuzumab ozogamicin (MylortargTM ), Bren growth factor a tissue inhibitor of metalloproteinases ; a tuximab vedotin ( AdcetrisTM ), 9°Y - labelled ibritumomab US 10 ,000 ,582 B2 12 tiuxetan ( ZevalinTM ), 1311 -labelled tositumoma ( BexxarTM ), tives ( e . g . , NSC 33410 ) , dolstatin 10 (NSC 376128 ) , may etc . Suitable antibodies for use in cancer treatment include , tansine (NSC 153858 ) , rhizoxin (NSC 332598 ) , paclitaxel but are not limited to , antibodies raised against tumor ( Taxol® ), Taxol® derivatives , docetaxel ( Taxotere® ) , thio associated antigens . Such antigens include, but are not colchicine (NSC 361792 ) , trityl cysterin , vinblastine sulfate , limited to , CD20 , CD30 , CD33, CD52 , EpCAM , CEA , 5 vincristine sulfate , natural and synthetic epothilones includ gpA33 , Mucins, TAG - 72 , CAIX , PSMA , Folate -binding ing but not limited to , eopthilone A , epothilone B , discod protein , Gangliosides ( e . g . , GD2, GD3, GM2, etc . ) , Le " , ermolide ; estramustine , nocodazole, and the like . VEGF, VEGFR , Integrin alpha - V -beta - 3 , Integrin alpha - 5 - Hormone modulators and steroids ( including synthetic beta - 1 , EGFR , ERBB2, ERBB3 , MET, IGFIR , EPHA3, analogs) that are suitable for use include , but are not limited TRAILR1, TRAILR2 , RANKL , FAP, Tenascin , etc . 10 to , adrenocorticosteroids, e . g . prednisone , dexamethasone , Biological response modifiers suitable for use include, but etc .; estrogens and pregestins , e . g . hydroxyprogesterone are not limited to , ( 1 ) inhibitors of tyrosine kinase (RTK ) caproate, medroxyprogesterone acetate, megestrol acetate, activity ; ( 2 ) inhibitors of serine / threonine kinase activity ; ( 3 ) estradiol, clomiphene , tamoxifen ; etc . ; and adrenocortical tumor -associated antigen antagonists , such as antibodies that suppressants , e . g . aminoglutethimide ; 17a - ethinylestradiol; bind specifically to a tumor antigen ; ( 4 ) apoptosis receptor 15 diethylstilbestrol, testosterone, fluoxymesterone , dromo agonists ; ( 5 ) interleukin - 2 ; (6 ) interferon - a ; ( 7 ) interferon - y ; stanolone propionate , testolactone , methylprednisolone, ( 8 ) colony - stimulating factors; ( 9 ) inhibitors of angiogen - methyl- testosterone , prednisolone, triamcinolone , chlorotri esis ; and ( 10 ) antagonists of tumor necrosis factor . anisene, hydroxyprogesterone, aminoglutethimide , estra Chemotherapeutic agents are non -peptidic ( i . e . , non -pro - mustine, medroxyprogesterone acetate , leuprolide , Fluta teinaceous) compounds that reduce proliferation of cancer 20 mide (Drogenil ) , Toremifene ( Fareston ) , and Zoladex® . cells , and encompass cytotoxic agents and cytostatic agents . Estrogens stimulate proliferation and differentiation , there Non - limiting examples of chemotherapeutic agents include fore compounds that bind to the estrogen receptor are used alkylating agents , nitrosoureas, antimetabolites , antitumor to block this activity . Corticosteroids may inhibit T cell antibiotics , plant (vinca ) alkaloids, and steroid hormones . proliferation . Agents that act to reduce cellular proliferation are known 25 Other chemotherapeutic agents include metal complexes , in the art and widely used . Such agents include alkylating e . g . cisplatin (cis - DDP ) , carboplatin , etc . ; ureas, e . g . agents , such as nitrogen mustards , nitrosoureas, ethylen - hydroxyurea ; and hydrazines , e . g . N -methylhydrazine ; epi imine derivatives , alkyl sulfonates, and triazenes , including , dophyllotoxin ; a topoisomerase inhibitor; procarbazine ; but not limited to , mechlorethamine , cyclophosphamide mitoxantrone ; leucovorin ; tegafur ; etc . Other anti - prolifera (CytoxanTM ) , melphalan (L - sarcolysin ), carmustine 30 tive agents of interest include immunosuppressants , e . g . (BCNU ) , lomustine (CCNU ) , semustine (methyl - CCNU ) , mycophenolic acid , thalidomide , desoxyspergualin , streptozocin , chlorozotocin , uracil mustard , chlormethine, azasporine, leflunomide , mizoribine, azaspirane ( SKF ifosfamide, chlorambucil, pipobroman , triethylen 105685 ); Iressa® (ZD 1839, 4 -( 3 -chloro - 4 - fluorophe emelamine , triethylenethiophosphoramine, busulfan , dacar nylamino )- 7 -methoxy - 6 - ( 3 - (4 -morpholinyl ) propoxy ) qui bazine , and temozolomide . 35 nazoline ); taxanes (including taxane derivatives ) ; etc . Antimetabolite agents include folic acid analogs , pyrimi- Utility dine analogs , purine analogs, and adenosine deaminase Sulfonated hyaluronic acid as described above can be inhibitors , including , but not limited to , cytarabine (CY - used in a variety of medical applications. Such applications TOSAR - U ) , cytosine arabinoside, fluorouracil ( 5 - FU ), include , e .g . , tissue engineering and drug delivery . As such , floxuridine (FudR ) , 6 - thioguanine , 6 -mercaptopurine 40 the present disclosure provides artificial tissues and drug (6 -MP ) , pentostatin , 5 - fluorouracil (5 - FU ) , methotrexate , delivery compositions comprising a subject sulfonated 10 - propargyl- 5 ,8 - dideazafolate (PDDF , CB3717 ), 5 , 8 - dide hyaluronic acid . azatetrahydrofolic acid (DDATHF ) , leucovorin , fludarabine For use in a medical application , such as tissue engineer phosphate , pentostatine, and gemcitabine . ing and drug delivery, a subject sulfonated hyaluronic acid Suitable natural products and their derivatives, ( e . g ., vinca 45 ( also referred to herein as " ethylsulfonated hyaluronic acid " alkaloids , antitumor antibiotics , enzymes , lymphokines , and or " ES -HYA ” ) can be used without further modification . epipodophyllotoxins ), include , but are not limited to , Ara - C , Alternatively, a subject ES -HyA can be further modified to paclitaxel ( Taxol® ), docetaxel ( Taxotere® ), deoxycoformy include 1, 2, 3, or more different moieties covalently or cin , mitomycin - C , L -asparaginase , azathioprine ; brequinar; non - covalently linked to the hyaluronic acid (HyA ) core . alkaloids, e . g . vincristine , vinblastine , vinorelbine , vin - 50 Implantable ES -HYA Material and Tissue Engineering desine , etc . ; podophyllotoxins , e . g . etoposide , teniposide , The present disclosure provides artificial tissue, e . g . , an etc . ; antibiotics , e . g . anthracycline , daunorubicin hydrochlo - implantable polymeric material , comprising a subject ES ride ( daunomycin , rubidomycin , cerubidine ) , idarubicin , HyA matrix . A subject implantable ES -HyA material can doxorubicin , epirubicin and morpholino derivatives , etc .; form a two - dimensional or a three - dimensional structure . A phenoxizone biscyclopeptides, e . g . dactinomycin ; basic gly - 55 subject implantable ES - HyA material can form a variety of copeptides , e . g . bleomycin ; anthraquinone glycosides, e . g . structures, including, but not limited to , surgical suture and plicamycin (mithramycin ); anthracenediones, e .g . mitoxan - ligature strands ; scaffolds and patches for soft and hard trone ; azirinopyrrolo indolediones, e . g . mitomycin ; macro - tissue regeneration ; guided bone regeneration (GBR ) ; cyclic immunosuppressants , e . g. cyclosporine , FK -506 guided tissue regeneration (GTR ); surgical meshes; gauze ( tacrolimus , prograf ) , rapamycin , etc . ; and the like . 60 for wound dressing ; artificial organs, artificial vessels ( e . g . , Other anti - proliferative cytotoxic agents are navelbene , artificial blood vessels , such as artificial arteries ; and the CPT- 11 , anastrazole , letrazole , capecitabine , reloxafine , like . A subject implantable ES -HyA material can comprise cyclophosphamide, ifosamide, and droloxafine . one or more active agents ( as described below ) . A subject Microtubule affecting agents that have antiproliferative implantable ES -HyA material can be coated onto a solid activity are also suitable for use and include , but are not 65 substrate comprising a second material, where suitable sec limited to , allocolchicine (NSC 406042 ), Halichondrin B ond materials include, but are not limited to , calcium phos (NSC 609395 ), colchicine (NSC 757 ), colchicine deriva phate ; titanium ; and the like . A subject implantable ES - HyA US 10 ,000 , 582 B2 13 14 material can be a tissue engineering scaffold . A subject ( e . g ., from about 5 Daltons to about 10 Daltons , from about implantable ES -HyA material can include living cells , e . g ., 10 Daltons to about 50 Daltons, from about 50 Daltons to stem cells ; bone cells; bone progenitors ; epithelial cells ; about 100 Daltons , from about 100 Daltons to about 500 neural cells ; neural progenitor cells ; endothelial cells; Daltons, from about 500 Daltons to about 1 kDa, or from muscle cells ; skin cells ; and the like . 5 about 1 kDa to about 5 kDa) . The present disclosure provides methods of treatment, Pharmacologically active agents useful for inclusion in a comprising implanting into an individual in need thereof a subject ES -HyA matrix include drugs acting at synaptic and subject implantable ES -HyA material. The present disclo - neuroeffector junctional sites ( cholinergic agonists , anticho sure provides a method of treating a subject having a linesterase agents , atropine , scopolamine , and related anti pathology characterized by diseased , damaged or loss of 10 muscarinic drugs, catecholamines and sympathomimetic tissue , the method comprising implanting subject implant- drugs , and adrenergic receptor antagonists ); drugs acting on able ES -HyA material at or near the diseased , damaged or the central nervous systems; autacoids ( drug therapy of loss tissue of the subject, thereby inducing the formation of inflammation ) ; drugs affecting renal function and electrolyte the tissue and treating the subject. metabolism ; cardiovascular drugs, drugs affecting gastroin Where a subject implantable ES - HyA material comprises 15 testinal function ; chemotherapy of neoplastic diseases ; living cells, a subject method provides for tissue regenera - drugs acting on the blood and the blood - forming organs; and tion . Where a subject implantable ES -HyA material com - hormones and hormone antagonists . Thus , the agents useful prises an active agent such as a bone morphogenetic protein , in the matrix composition include , but are not limited to a subject method provides for bone growth /bone regenera - anti - infectives such as antibiotics and antiviral agents ; anal tion . Where a subject implantable ES -HyA material com - 20 gesics and analgesic combinations; local and general anes prises an active agent such as a clotting factor, a subject thetics ; anorexics; antiarthritics ; antiasthmtic agents ; anti method provides for wound healing . Where a subject convulsants ; antidepressants ; antihistamines; anti implantable ES -HyA material comprises an active agent inflammatory agents ; antinauseants; antimigrane agents ; such as an antibiotic , a subject method provides for wound antineoplastics; antipruritics ; antipsychotics ; antipyretics ; healing. 25 antispasmodics ; cardiovascular preparations ( including cal The present disclosure provides a method of inducing ex cium channel blockers , beta -blockers , beta -agonists and vivo formation of a tissue, the method comprising : seeding antiarrythmics ) ; antihypertensives ; diuretics; vasodilators ; a subject implantable ES -HyA matrix with cells in a medium central nervous system stimulants , cough and cold prepara suitable for proliferation , differentiation and /or migration of tions ; decongestants ; diagnostics ; hormones; bone growth said cells, thereby inducing the formation of the tissue. The 30 stimulants and bone resorptioninhibitors ; immunosuppres present disclosure provides a method of inducing in vivo sives ; muscle relaxants ; psychostimulants ; sedatives ; tran formation of a tissue, the method comprising implanting a quilizers ; proteins , peptides , and fragments thereof (whether subject implantable ES - HyA material in a subject, where the naturally occurring , chemically synthesized or recombi implantable ES -HyA material comprises a subject ES -HyA nantly produced ); and nucleic acid molecules (polymeric matrix and living cells within the ES -Hy A matrix , wherein 35 forms of two or more nucleotides , either ribonucleotides said implanting induces the formation of the tissue. (RNA ) or deoxyribonucleotides (DNA ) including double Drug Delivery and single -stranded molecules and supercoiled or condensed A subject ES -HyA can be used as a drug delivery matrix ; molecules , gene constructs , expression vectors , plasmids , e . g . , a subject ES -HyA can form a matrix . For example , an antisense molecules and the like . active agent is encapsulated within a subject ES - HYA 40 Small Molecule Drugs matrix . An active agent can be embedded within a subject Any of a variety of small molecule active agents (“ drugs ” ) ES - HyA matrix . An active agent can be non -covalently can be included in a subject ES -HyA matrix . Non - limiting associated with a subject ES -HyA matrix . An active agent examples include lipid - regulating agents ; sex hormones ; can be covalently linked to a subject ES -HyA matrix . androgenic agents ; antihypertensive agents ; anti - diabetic Thus, the present disclosure provides an ES -Hy drug 45 agents ; anti - viral agents ; and active agents of any of the delivery composition comprising: a ) a subject ES - HYA other below - listed categories . matrix ; and b ) an active agent associated with , embedded Lipid - regulating agents that are generally classified as with , or encapsulated within , the ES - Hy matrix . hydrophobic include HMG CoA reductase inhibitors such as The present disclosure provides treatment methods com atorvastatin , simvastatin , fluvastatin , pravastatin , lovastatin , prising administering to an individual in need thereof a 50 cerivastatin , rosuvastatin , and pitavastatin , as well as other subject ES -HYA drug delivery composition , wherein the lipid - lowering ( “ antihyperlipidemic ” ) agents such as beza active agent is present in the ES -HyA drug delivery com fibrate , beclobrate , binifibrate , ciprofibrate , clinofibrate , clo position in an amount effective to treat a disease or disorder fibrate , clofibric acid , ezetimibe , etofibrate , fenofibrate , in the individual. fenofibric acid , gemfibrozil, nicofibrate , pirifibrate , probu Active Agents 55 col, ronifibrate , simfibrate , and theofibrate . Active agents that can be included in a subject ES -HyA Sex hormones include , e . g . , progestins ( progestogens) , matrix include , but are not limited to , small molecule drugs, estrogens, and combinations thereof. Progestins include peptides , microRNAs (miRNA ) , and interfering RNAs . acetoxypregnenolone, allylestrenol, anagestone acetate , Small molecule drugs include drugs having a molecular chlormadinone acetate , cyproterone , cyproterone acetate , weight of from about 5 Daltons to about 50 kDaltons (kDa ) 60 desogestrel, dihydrogesterone , dimethisterone , ethisterone ( e . g . , from about 5 Daltons to about 10 Daltons, from about ( 17a - ethinyltestosterone ) , ethynodiol diacetate , fluroge 10 Daltons to about 50 Daltons, from about 50 Daltons to stone acetate , gestadene , hydroxyprogesterone , hydroxypro about 100 Daltons , from about 100 Daltons to about 500 gesterone acetate , hydroxyprogesterone caproate , Daltons, from about 500 Daltons to about 1 kDa, from about hydroxymethylprogesterone , hydroxymethylprogesterone 1 kDa to about 5 kDa , from about 5 kDa to about 10 kDa, 65 acetate , 3 -ketodesogestrel , levonorgestrel, lynestrenol, from about 10 kDa to about 25 kDa, or from about 25 kDa medrogestone , medroxyprogesterone acetate , megestrol, to about 50 kDa ) , or from about 5 Daltons to about 5 kDa megestrol acetate , melengestrol acetate , norethindrone, US 10 ,000 ,582 B2 15 16 norethindrone acetate , norethisterone, norethisterone Additional suitable active agents include: acetate , norethynodrel, norgestimate , norgestrel, norg antianti - inflammatory agents and non -opioid analgesics, such estrienone , normethisterone, progesterone , and trimgestone . as aloxiprin , auranofin , azapropazone, azathioprine , beno Also included within this general class are estrogens , e . g . : rylate , butorphenol, capsaicin , celecoxib , diclofenac , estradiol (i . e ., 1 ,3 , 5 -estratriene -3 , 17ß -diol , or “ 17B -estra - 5 diflunisal, esonarimod , etodolac, fenbufen , fenoprofen cal diol” ) and its esters , including estradiol benzoate , valerate , cium , flurbiprofen , ibuprofen , indomethacin , ketoprofen , ketorolac , leflunomide , meclofenamic acid , mefenamic acid , cypionate , heptanoate , decanoate , acetate and diacetate ; nabumetone , naproxen , novantrone , oxaprozin , oxyphenb 2 -Methoxyestradiol ; 4 - Hydroxyestradiol; 17a -estradiol ; utazone , parecoxib , phenylbutazone , piclamilast, piroxicam , ethinylestradiol ( i. e ., 17a - ethinylestradiol) and esters and 10 rofecoxib , ropivacaine, sulindac, tetrahydrocannabinol , tra ethers thereof, including ethinylestradiol 3 -acetate and ethi madol, tromethamine , valdecoxib , and ziconotide , as well as nylestradiol 3 -benzoate ; estriol and estriol succinate ; poly the urinary analgesics phenazopyridine and tolterodine ; estrol phosphate ; estrone and its esters and derivatives , anti - angina agents , such as mibefradil , refludan , nahne including estrone acetate , estrone sulfate , and piperazine fene, carvedilol, cromafiban , lamifiban , fasudil , ranolazine , estrone sulfate ; quinestrol; mestranol; and conjugated 15 tedisamil , nisoldipine, and tizanidine; equine estrogens . In many contexts, e . g ., in female contra antihelminthics , such as albendazole , bephenium ception and in hormone replacement therapy (HRT ) , a hydroxynaphthoate , cambendazole, dichlorophen , ivermec combination of a progestin and estrogen is used , e . g ., tin , mebendazole , oxamniquine , oxfendazole , oxantel progesterone and 17B - estradiol. For HRT, an androgenic embonate , praziquantel, pyrantel embonate and thiabenda agent may be advantageously included as well. Androgenic 20 zole ; agents for this purpose include, for example , dehydroepi anti - arrhythmic agents , such as amiodarone, disopyra androsterone (DHEA ; also termed " prasterone” ) , sodium mide, flecainide acetate and quinidine sulfate ; dehydroepiandrosterone sulfate , 4 - dihydrotestosterone anti - asthma agents, such as zileuton , zafirlukast, terbuta (DHT ; also termed “ stanolone ” ) , and testosterone , and phar - line sulfate , montelukast, and albuterol; maceutically acceptable esters of testosterone and 4 - dihy - 25 anti - bacterial agents , such as alatrofloxacin , azithromy drotestosterone , typically esters formed from the hydroxyl cin , baclofen , benethamine penicillin , cinoxacin , ciprofloxa group present at the C - 17 position , including , butnot limited cin , clarithromycin , clofazimine, cloxacillin , demeclocy to , the enanthate , propionate , cypionate , phenylacetate , cline , dirithromycin , doxycycline , erythromycin , acetate , isobutyrate , buciclate , heptanoate , decanoate , unde - ethionamide , furazolidone , grepafloxacin , imipenem , levo canoate , caprate and isocaprate esters . 30 floxacin , lorefloxacin , moxifloxacin , nalidixic acid , nitro Other androgenic agents include , but are not limited to , furantoin , norfloxacin , ofloxacin , rifampicin , rifabutine , rifa androsterone, androsterone acetate , androsterone propi pentine , sparfloxacin , spiramycin , sulphabenzamide , onate, androsterone benzoate , androstenediol, androstene sulphadoxine , sulphamerazine , sulphacetamide, sulphadiaz diol- 3 - acetate , androstenediol- 17 -acetate , androstenediol- 3 , ine, sulphafurazole, sulphamethoxazole , sulphapyridine , tet 17 - diacetate , androstenediol - 17 - benzoate , androstenediol- 3 - 35 racycline , trimethoprim , trovafloxacin , and vancomycin ; acetate - 17 -benzoate , androstenedione , ethylestrenol, anti - cancer agents and immunosuppressants, such as ali oxandrolone , nandrolone phenpropionate , nandrolone tretinoin , aminoglutethimide , amsacrine , anastrozole , aza decanoate , nandrolone furylpropionate , nandrolone cyclo - thioprine , bexarotene, bicalutamide , biricodar, bisantrene , hexane - propionate , nandrolone benzoate , nandrolone cyclo - busulfan , camptothecin , candoxatril, capecitabine, cytara hexanecarboxylate , stanozolol, dromostanolone , and dromo- 40 bine , chlorambucil , cyclosporin , dacarbazine , decitabine, stanolone propionate . ellipticine, estramustine, etoposide, gemcitabine , irinotecan , Antihypertensive agents include , without limitation , lasofoxifene , letrozole , lomustine, melphalan , mercaptopu amlodipine , benazepril, benidipine , candesartan , captopril, rine , methotrexate , mitomycin , mitotane, mitoxantrone , carvedilol, darodipine, dilitazem , diazoxide , doxazosin , mofetil , mycophenolate , nebivolol, nilutamide , paclitaxel , enalapril , epleronone , eposartan , felodipine , fenoldopam , 45 palonosetron , procarbazine, ramipril , rubitecan , sirolimus, fosinopril , guanabenz , iloprost, irbesartan , isradipine , lercar - tacrolimus , tamoxifen , teniposide , testolactone, thalidomide , dinipine, lisinopril, losartan , minoxidil, nebivolol, nicar - tirapazamine , topotecan , toremifene citrate , vitamin A , vita dipine, nifedipine , nimodipine, nisoldipine , omapatrilat, min A derivatives, and zacopride ; phenoxybenzamine , prazosin , quinapril, reserpine , semo - anti- coagulants and other agents for preventing and treat tiadil , sitaxsentan , terazosin , telmisartan , and valsartan . 50 ing stroke, such as cilostazol, citicoline , clopidogrel, cro Anti- diabetic agents include , by way of example , aceto - mafiban , dexanabinol, dicumarol, dipyridamole , nicouma hexamide , chlorpropamide, ciglitazone , farglitazar, gliben - lone, oprelvekin , perindopril erbumine, phenindione , clamide , gliclazide, glipizide , glucagon , glyburide , ramipril , repinotan , ticlopidine , tirofiban , and heparin , glymepiride, miglitol, pioglitazone , nateglinide , pimage including heparin salts formed with organic or inorganic dine , repaglinide, rosiglitazone , tolazamide, tolbutamide , 55 bases , and low molecular weight heparin , i. e ., heparin frag triampterine , and troglitazone. ments generally having a weight average molecular weight Antiviral agents that can be included in a subject ES -Hy in the range of about 1000 to about 10 , 000 D and exempli matrix include the antiherpes agents acyclovir , famciclovir , fied by enoxaparin , dalteparin , danaproid , gammaparin , foscarnet, ganciclovir, idoxuridine , sorivudine, trifluridine , nadroparin , ardeparin , tinzaparin , certoparin , and reviparin ; valacyclovir, and vidarabine, and other antiviral agents such 60 anti -diabetics , such as acetohexamide , chlorpropamide , as abacavir, amantadine, amprenavir , delviridine , didanos farglitazar, glibenclamide , gliclazide , glipizide, glimepiride , ine, efavirenz , indinavir, interferon alpha, lamivudine , nel miglitol, nateglinide , pimagedine , pioglitazone, repaglinide , finavir , nevirapine , ribavirin , rimantadine, ritonavir, saqui- rosiglitazone , tolazamide , tolbutamide, troglitazone , and navir , stavudine , tipranavir , valganciclovir , zalcitabine , and voglibose ; zidovudine ; and other antiviral agents such as abacavir , 65 anti - epileptics, such as beclamide, carbamazepine, clon indinavir, interferon alpha , nelfinavir, ribavirin , rimantadine , azepam , ethotoin , felbamate , fosphenytoin , lamotrigine , tipranavir , ursodeoxycholic acid , and valganciclovir. methoin , methsuximide, methylphenobarbitone , oxcarba US 10 ,000 , 582 B2 17 18 zepine , paramethadione , phenacemide, phenobarbitone , nifedipine , nicardipene, felodipine, isradipine , nimodipine , phenytoin , phensuximide , primidone , sulthiame, tiagabine , amlodipine and diltiazem ; beta - blockers such as acebutolol, topiramate , valproic acid , and vigabatrin ; alprenolol, atenolol, labetalol, metoprolol, nadolol, oxypre anti -fungal agents , such as amphotericin , butenafine , nolol, pindolol, propafenone , propranolol, esmolol, sotalol, butoconazole nitrate , clotrimazole , econazole nitrate , flu - 5 timolol, and acebutolol; antiarrhythmics such as moricizine , conazole , flucytosine , griseofulvin , itraconazole , ketocon - dofetilide , ibutilide , nesiritide , procainamide , quinidine, azole , miconazole , natamycin , nystatin , sulconazole nitrate , disopyramide , lidocaine , phenytoin , tocainide, mexiletine , oxiconazole , terbinafine , terconazole , tioconazole and unde - flecainide , encainide, bretylium and amiodarone ; cardiopro cenoic acid ; tective agents such as dexrazoxane and leucovorin ; vasodi anti - gout agents , such as allopurinol, probenecid and 10 lators such as nitroglycerin ; diuretic agents such as azeta sulphin - pyrazone ; zolamide, amiloride , bendroflumethiazide, bumetanide , antihistamines and allergy medications, such as acrivas - chlorothiazide , chlorthalidone , ethacrynic acid , furosemide , tine , astemizole , chlorpheniramine, cinnarizine , cetirizine, hydrochlorothiazide , metolazone, nesiritide, spironolactone, clemastine , cyclizine, cyproheptadine, desloratadine, dex and triamterine ; and miscellaneous cardiovascular drugs chlorpheniramine, dimenhydrinate , diphenhydramine , epi- 15 such as monteplase and corlopam ; nastine , fexofenadine , flunarizine , loratadine , meclizine , corticosteroids , such as beclomethasone , betamethasone , mizolastine , oxatomide , and terfenadine ; budesonide , cortisone , desoxymethasone , dexamethasone , anti -malarials , such as amodiaquine , chloroquine, chlo fludrocortisone , flunisolide, fluocortolone, fluticasone pro rproguanil, halofantrine, mefloquine , proguanil, pyrimeth - pionate , hydrocortisone , methylprednisolone , prednisolone , amine and quinine sulfate ; 20 prednisone and triamcinolone ; agents for treating headaches, including anti -migraine erectile dysfunction drugs, such as apomorphine , phen agents , such as almotriptan , butorphanol, dihydroergot - tolamine , and vardenafil ; amine, dihydroergotamine mesylate , eletriptan , ergotamine, gastrointestinal agents , such as alosetron , bisacodyl , frovatriptan , methysergide, naratriptan , pizotyline , rizatrip - cilansetron , cimetidine , cisapride , diphenoxylate , domperi tan , sumatriptan , tonaberstat, and zolmitriptan ; 25 done, esomeprazole , famotidine, granisetron , lansoprazole , anti -muscarinic agents , such as atropine , benzhexol, loperamide, mesalazine , nizatidine, omeprazole , ondanse biperiden , ethopropazine, hyoscyamine , mepenzolate bro - tron , prantoprazole , rabeprazole sodium , ranitidine , risperi mide , oxyphencyclimine , scopolamine , and tropicamide; done, sulphasalazine , and tegaserod ; anti -protozoal agents , such as atovaquone, benznidazole , keratolytics , such as such as acetretin , calcipotriene , cal clioquinol, decoquinate , diiodohydroxyquinoline, dilox - 30 cifediol, calcitriol, cholecalciferol, ergocalciferol, etretinate , anide furoate , dinitolmide , furazolidone , metronidazole , retinoids , targretin , and tazarotene ; nimorazole , nitrofirazone, ornidazole and tinidazole ; lipid regulating agents, such as atorvastatin , bezafibrate , anti - thyroid agents , such as carbimazole , paricalcitol, and cerivastatin , ciprofibrate , clofibrate , ezetimibe , fenofibrate , propylthiouracil ; fluvastatin , gemfibrozil , pitavastatin , pravastatin , probucol, anti- tussives , such as benzonatate ; 35 rosuvastatin , and simvastatin ; anxiolytics , sedatives , and hypnotics, such as alprazolam , muscle relaxants , such as cyclobenzaprine , dantrolene amylobarbitone, barbitone , bentazepam , bromazepam , bro sodium and tizanidine HCl; mperidol, brotizolam , butobarbitone, carbromal, chlordiaz - agents to treat neurodegenerative diseases , including epoxide , chlormethiazole , chlorpromazine , chlorprothixene , active agents for treating Alzheimer' s disease such as aka clonazepam , clobazam , clotiazepam , clozapine, dexmethyl- 40 tinol, donezepil, donepezil hydrochloride, dronabinol, gal phenidate ( d - threo -methylphenidate ) diazepam , droperidol, antamine , neotrofin , rasagiline , physostigmine , physostig ethinamate , flunanisone, flunitrazepam , triflupromazine, flu - mine salicylate , propentoffyline, quetiapine , rivastigmine , penthixol decanoate , fluphenazine, flurazepam , gabapentin , tacrine , tacrine hydrochloride, thalidomide, and xaliproden ; gaboxadol, y -hydroxybutyrate , haloperidol, lamotrigine , active agents for treating Huntington ' s Disease , such as lorazepam , lormetazepam , medazepam , meprobamate , 45 fluoxetine and carbamazepine ; anti -parkinsonism drugs use mesoridazine , methaqualone , methylphenidate, midazolam , ful herein include amantadine , apomorphine , bromocriptine , modafinil, molindone , nitrazepam , olanzapine, oxazepam , entacapone, levodopa ( particularly a levodopa/ carbidopa pentobarbitone , perphenazine pimozide , pregabalin , combination ), lysuride , pergolide , pramipexole , rasagiline , prochlorperazine , pseudoephedrine , quetiapine, rispiridone, riluzole , ropinirole , selegiline , sumanirole , tolcapone , tri sertindole , siramesine , sulpiride, sunepitron , temazepam , 50 hexyphenidyl, and trihexyphenidyl hydrochloride ; and thioridazine , triazolam , zaleplon , zolpidem , and zopiclone; active agents for treating ALS such as the anti - spastic agents appetite suppressants , anti- obesity drugs and drugs for baclofen , diazemine , and tizanidine ; treatment of eating disorders , such as amphetamine , bro - nitrates and other anti - anginal agents , such as amyl mocriptine , dextroamphetamine , diethylpropion , lintitript, nitrate , glyceryl trinitrate , isosorbide dinitrate , isosorbide mazindol, methamphetamine , orlistat, phentermine , and 55 mononitrate and pentaerythritol tetranitrate ; topiramate ; neuroleptic drugs , including antidepressant drugs , anti cardiovascular drugs, including: angiotensin converting manic drugs , and antipsychotic agents , wherein antidepres enzyme (ACE ) inhibitors such as enalapril , ramipril , perin sant drugs include ( a ) the tricyclic antidepressants such as dopril erbumine , 1 -carboxymethyl -3 - 1 -carboxy - 3 -phenyl amoxapine, amitriptyline, clomipramine , desipramine , dox ( 1S ) -propylamino - 2 , 3 , 4 , 5 - tetrahydro - 1H - ( 3S ) - 1 -ben - 60 epin , imipramine , maprotiline , nortriptyline , protriptyline , zazepine - 2 - one , 3 - ( 5 - amino - 1 - carboxy - 1S -pentyl ) amino - 2 , and trimipramine, ( b ) the serotonin reuptake inhibitors cit 3 , 4 , 5 - tetrahydro - 2 - oxo -3S - 1H - 1 -benzazepine - lacetic acid or alopram , fluoxetine , fluvoxamine , paroxetine , sertraline , and 3 - ( 1 - ethoxycarbonyl- 3 -phenyl - ( 1S ) -propylamino ) - 2 , 3 , 4 , 5 - venlafaxine, ( c ) monoamine oxidase inhibitors such as tetrahydro - 2 -oxo - ( - 3S ) - benzazepi acid monohydrochloride ; phenelzine , tranylcypromine, and ( - ) - selegiline , and ( d ) cardiac glycosides and cardiac inotropes such as amrinone , 65 other antidepressants such as aprepitant, bupropion , dulox digoxin , digitoxin , enoximone , lanatoside C , medigoxin , and etine , gepirone, igmesine , lamotrigine , maprotiline , mian milrinone ; calcium channel blockers such as verapamil, serin , mirtazapine, nefazodone, rabalzotan , sunepitron , tra US 10 , 000 , 582 B2 19 20 zodone and venlafaxine , and wherein antimanic and doephedrine, pyridostigmine , rabeprazole , raloxifene, repa antipsychotic agents include ( a ) phenothiazines such as glinide , rifabutine , rifapentine , rimexolone , ritanovir , riza acetophenazine , acetophenazine maleate , chlorpromazine , triptan , rofecoxib , rosiglitazone , Saquinavir, sertraline , chlorpromazine hydrochloride , fluphenazine , fluphenazine sibutramine , sildenafil citrate , simvastatin , sirolimus, hydrochloride , fluphenazine enanthate , fluphenazine 5 spironolactone , sumatriptan , tacrine , tacrolimus , tamoxifen , decanoate , mesoridazine, mesoridazine besylate , per - tamsulosin , targretin , tazarotene , telmisartan , teniposide , phenazine , thioridazine, thioridazine hydrochloride , triflu - terbinafine , terazosin , tetrahydrocannabinol, tiagabine, ticlo operazine , and trifluoperazine hydrochloride , ( b ) thioxan - pidine , tirofiban , tizanidine , topiramate , topotecan , tore thenes such as chlorprothixene , thiothixene , and thiothixene mifene , tramadol, tretinoin , troglitazone , trovafloxacin , ubi hydrochloride , and ( c ) other heterocyclic drugs such as 10 decarenone , valsartan , venlafaxine , verteporfin , vigabatrin , carbamazepine , clozapine , droperidol, haloperidol, haloperi- vitamin A , vitamin D , vitamin E , vitamin K , zafirlukast , dol decanoate , loxapine succinate , molindone , molindone zileuton , zolmitriptan , zolpidem , zopiclone, and combina hydrochloride , olanzapine , pimozide, quetiapine , risperi tions thereof. done , and sertindole ; Hydrophilic Active Agents nutritional agents , such as calcitriol, carotenes , dihydrot - 15 Non - limiting examples of hydrophilic active agents achysterol, essential fatty acids, non - essential fatty acids , include , without limitation , acarbose , acyclovir, acetyl cys phytonadiol , vitamin A , vitamin B2, vitamin D , vitamin E teine , acetylcholine chloride , alatrofloxacin , alendronate , and vitamin K ; alglucerase , amantadine hydrochloride , ambenomium , ami opioid analgesics , such as alfentanil , apomorphine , fostine , amiloride hydrochloride, aminocaproic acid , buprenorphine , butorphanol, codeine , dextropropoxyphene , 20 amphotericin B , antihemophilic factor (human ), antihemo diamorphine, dihydrocodeine , fentanyl, hydrocodone , philic factor (porcine ) , antihemophilic factor ( recombinant ) , hydromorphone , levorphanol, meperidine, meptazinol, aprotinin , asparaginase , atenolol , atracurium besylate , atro methadone, morphine, nalbuphine , oxycodone , oxymor - pine , azithromycin , aztreonam , BCG vaccine , bacitracin , phone , pentazocine , propoxyphene , sufentanil , and trama - becaplermin , belladona , bepridil hydrochloride , bleomycin dol; and 25 sulfate , calcitonin human , calcitonin salmon , carboplatin , stimulants , including active agents for treating narco - capecitabine , capreomycin sulfate , cefamandole nafate , lepsy , attention deficit disorder ( ADD ) and attention deficit cefazolin sodium , cefepime hydrochloride , cefixime, cefo hyperactivity disorder (ADHD ) , such as amphetamine , dex nicid sodium , cefoperazone , cefotetan disodium , cefo amphetamine , dexfenfluramine , fenfluramine, mazindol, taxime, cefoxitin sodium , ceftizoxime, ceftriaxone , cefurox methylphenidate ( including d - threo -methylphenidate , or 30 ime axetil , cephalexin , cephapirin sodium , cholera vaccine , " dexmethylphenidate , ” as well as racemic d , l - threo -methyl - chorionic gonadotropin , cidofovir , cisplatin , cladribine , cli phenidate ) , modafinil, pemoline , and sibutramine. dinium bromide , clindamycin and clindamycin derivatives, Hydrophobic Active Agents ciprofloxacin , clodronate , colistimethate sodium , colistin Non - limiting examples of hydrophobic active agents sulfate , corticotropin , cosyntropin , cromolyn sodium , cyt include , but are not limited to , acetretin , acetyl coenzyme Q , 35 arabine , dalteparin sodium , danaparoid , deferoxamine , albendazole , albuterol, aminoglutethimide , amiodarone , denileukin diftitox , desmopressin , diatrizoate meglumine amlodipine , amphetamine, amphotericin B , atorvastatin , and diatrizoate sodium , dicyclomine , didanosine, dirithro atovaquone , azithromycin , baclofen , beclomethasone , mycin , dopamine hydrochloride , dornase alpha , doxacurium benazepril, benzonatate , betamethasone , bicalutanide, chloride , doxorubicin , etidronate disodium , enalaprilat , budesonide , bupropion , busulfan , butenafine , calcifediol, 40 enkephalin , enoxaparin , enoxaprin sodium , ephedrine, epi calcipotriene , calcitriol, camptothecin , candesartan , cap - nephrine , epoetin alpha , erythromycin , esmolol hydrochlo saicin , carbamezepine , carotenes , celecoxib , cerivastatin , ride , factor IX , famciclovir , fludarabine, fluoxetine , fosca cetirizine , chlorpheniramine, cholecalciferol, cilostazol, met sodium , ganciclovir , granulocyte colony stimulating cimetidine, cinnarizine , ciprofloxacin , cisapride , clarithro factor, granulocyte- macrophage stimulating factor, recom mycin , clemastine , clomiphene , clomipramine , clopidogrel, 45 binant human growth hormone , bovine growth hormine, codeine , coenzyme Q10 , cyclobenzaprine , cyclosporin , gentamycin , glucagon , glycopyrolate , gonadotropin releas danazol, dantrolene, dexchlorpheniramine , diclofenac , ing hormone and synthetic analogs thereof, gonadorelin , dicumarol, digoxin , dehydroepiandrosterone, dihydroergot grepafloxacin , haemophilus B conjugate vaccine, hepatitis A amine , dihydrotachysterol, dirithromycin , donezepil , efa virus vaccine inactivated , hepatitis B virus vaccine inacti virenz , eposartan , ergocalciferol, ergotamine , essential fatty 50 vated , heparin sodium , indinavir sulfate , influenza virus acid sources, estradiol, etodolac , etoposide , famotidine , vaccine, interleukin - 2 , interleukin - 3 , insulin - human , insulin fenofibrate, fentanyl, fexofenadine, finasteride , fluconazole , lispro , insulin procine , insulin NPH , insulin aspart , insulin flurbiprofen , fluvastatin , fosphenytoin , frovatriptan , fura glargine , insulin detemir , interferon alpha , interferon beta , zolidone, gabapentin , gemfibrozil , glibenclamide , glipizide, ipratropium bromide , ifosfamide, Japanese encephalitis glyburide , glimepiride , griseofulvin , halofantrine , ibupro - 55 virus vaccine , lamivudine , leucovorin calcium , leuprolide fen , irbesartan , irinotecan , isosorbide dinitrate , isotretinoin , acetate , levofloxacin , lincomycin and lincomycin deriva itraconazole , ivermectin , ketoconazole, ketorolac , lam tives , lobucavir , lomefloxacin , loracarbef, mannitol, measles otrigine, lansoprazole , leflunomide, lisinopril, loperamide , virus vaccine, meningococcal vaccine, menotropins, mepen loratadine , lovastatin , L - thyroxine , lutein , lycopene , zolate bromide , mesalamine, methenamine , methotrexate , medroxyprogesterone, mifepristone , mefloquine , megestrol 60 methscopolamine , metformin hydrochloride , metoprolol, acetate , methadone , methoxsalen , metronidazole , micon - mezlocillin sodium , mivacurium chloride , mumps viral vac azole , midazolam , miglitol, minoxidil, mitoxantrone, mon cine , nedocromil sodium , neostigmine bromide , neostig telukast , nabumetone , nalbuphine , naratriptan , nelfinavir , mine methyl sulfate , neurontin , norfloxacin , octreotide nifedipine , nisoldipine , nilutanide, nitro furantoin , nizati - acetate , ofloxacin , olpadronate , oxytocin , pamidronate diso dine , omeprazole , oprevelkin , oxaprozin , paclitaxel, para - 65 dium , pancuronium bromide , paroxetine , perfloxacin , pent calcitol, paroxetine , pentazocine , pioglitazone , pizofetin , amidine isethionate , pentostatin , pentoxifylline, penciclovir , pravastatin , prednisolone, probucol, progesterone , pseu pentagastrin , phentolamine mesylate , phenylalanine , phys US 10 , 000 , 582 B2 22 ostigmine salicylate , plague vaccine , piperacillin sodium , roglobulin , antithrombin III, factor I ( fibrinogen ) , factor II platelet derived growth factor, pneumococcal vaccine poly (prothrombin ) , factor III ( tissue prothrombin ), factor V valent, poliovirus vaccine ( inactivated ), poliovirus vaccine (proaccelerin ), factor VII ( proconvertin ), factor VIII ( anti live (OPV ) , polymyxin B sulfate , pralidoxime chloride, hemophilic globulin or AHG ), factor IX ( Christmas factor, pramlintide , pregabalin , propafenone, propenthaline bro - 5 plasma thromboplastin component or PTC ) , factor X (Stu mide , pyridostigmine bromide , rabies vaccine , risedronate , art -Power factor ) , factor XI (plasma thromboplastin ante ribavirin , rimantadine hydrochloride, rotavirus vaccine, sal cedent or PTA ), factor XII (Hageman factor ), heparin cofac meterol xinafoate , sincalide, small pox vaccine, solatol, tor II , kallikrein , plasmin , plasminogen , prekallikrein , somatostatin , sparfloxacin , spectinomycin , stavudine , strep - protein C , protein S , and thrombomodulin and combinations tokinase , streptozocin , suxamethonium chloride , tacrine 10 thereof hydrochloride, terbutaline sulfate , thiopeta , ticarcillin , RNAi tiludronate , timolol, tissue type plasminogen activator, Interfering RNA (RNAi ) include , e . g ., antisense RNA , a TNFR :Fc , TNK - UPA , trandolapril, trimetrexate gluconate , ribozyme, an RNAi and an siRNA . RNAi fragments , par trospectomycin , trovafloxacin , tubocurarine chloride, tumor ticularly double - stranded ( ds ) RNAi, can be used to inhibit necrosis factor, typhoid vaccine live , urea , urokinase , van - 15 gene expression . One approach well known in the art for comycin , valacyclovir , valsartan , varicella virus vaccine inhibiting gene expression is short interfering RNA ( siRNA ) live , vasopressin and vasopressin derivatives, vecuronium mediated gene silencing , where the level of expression bromide , vinblastine , vincristine , vinorelbine , vitamin B12 , product of a target gene is reduced by specific double warfarin sodium , yellow fever vaccine , zalcitabine , zanami- stranded siRNA nucleotide sequences that are complemen vir, zolendronate , zidovudine, and combinations thereof. 20 tary to at least a 19 - 25 nucleotide long segment ( e . g ., a 20 - 21 Polypeptide Agents nucleotide sequence ) of the target gene transcript, including Peptidyl drugs include therapeutic peptides and proteins the 5 ' untranslated (UT ) region , the ORF , or the 3 'UT region . per se , whether naturally occurring , chemically synthesized . In some embodiments , short interfering RNAs are about recombinantly produced , and /or produced by biochemical 19 - 25 nt in length . See , e . g . , PCT applications WO0 /44895 , ( e . g ., enzymatic ) fragmentation of larger molecules, and 25 WO99 /32619 , W001 / 75164 , W001/ 92513, W001/ 29058 , may contain the native sequence or an active fragment W001 /89304 , WO02 / 16620 , and WO02 / 29858 ; and U . S . thereof. Specific peptidyl drugs include , without limitation , Patent Publication No . 20040023390 for descriptions of the peptidyl hormones activin , amylin , angiotensin , atrial siRNA technology . The siRNA can be encoded by a nucleic natriuretic peptide (ANP ) , calcitonin , calcitonin gene- re - acid sequence , and the nucleic acid sequence can also lated peptide , calcitonin N - terminal flanking peptide , ciliary 30 include a promoter. The nucleic acid sequence can also neurotrophic factor (CNTF ) , corticotropin ( adrenocorti - include a polyadenylation signal. In some embodiments , the cotropin hormone, ACTH ) , corticotropin -releasing factor polyadenylation signal is a synthetic minimal polyade (CRF or CRH ) , epidermal growth factor ( EGF ) , follicle - nylation signal. stimulating hormone (FSH ) , gastrin , gastrin inhibitory pep Target genes include any gene encoding a target gene tide (GIP ) , gastrin - releasing peptide , gonadotropin - releasing 35 product (RNA or protein ) that is deleterious ( e . g . , patho factor (GnRF or GNRH ), growth hormone releasing factor logical ); a target gene product that is malfunctioning ; a (GRF , GRH ) , human chorionic gonadotropin ( hCH ), inhibin target gene product. Target gene products include , but are A , inhibin B , insulin , luteinizing hormone (LH ) , luteinizing not limited to , huntingtin ; hepatitis C virus ; human immu hormone - releasing hormone (LHRH ) , a - melanocyte - stimu- nodeficiency virus; amyloid precursor protein ; tau ; a protein lating hormone , B -melanocyte - stimulating hormone , y -mel - 40 that includes a polyglutamine repeat; a herpes virus ( e . g . , anocyte - stimulating hormone, melatonin , motilin , oxytocin varicella zoster ); any pathological virus ; and the like . (pitocin ) , pancreatic polypeptide, parathyroid hormone siRNA is useful for treating a variety of disorders and ( PTH ) , placental lactogen , prolactin (PRL ) , prolactin - re - conditions, including, but not limited to , neurodegenerative lease inhibiting factor (PIF ) , prolactin - releasing factor diseases, e . g . , a trinucleotide - repeat disease , such as a dis ( PRF ) , secretin , somatotropin ( growth hormone , GH ) , soma- 45 ease associated with polyglutamine repeats , e . g . , Hunting tostatin ( SIF , growth hormone - release inhibiting factor, ton ' s disease , spinocerebellar ataxia , spinal and bulbar mus GIF ) , thyrotropin ( thyroid - stimulating hormone , TSH ) , thy - cular atrophy (SBMA ) , dentatorubropallidoluysian atrophy rotropin - releasing factor ( TRH or TRF ) , thyroxine, vasoac - (DRPLA ) , etc . ; an acquired pathology ( e . g . , a disease or tive intestinal peptide (VIP ), and vasopressin . Other peptidyl syndrome manifested by an abnormal physiological, bio drugs are the cytokines , e . g . , colony stimulating factor 4 , 50 chemical, cellular , structural, or molecular biological state ) heparin binding neurotrophic factor ( HBNF ) , interferon - a , such as a viral infection , e . g . , hepatitis that occurs or may interferon a - 2a , interferon a - 2b , interferon a - n3 , interferon - occur as a result of an HCV infection , acquired immunode B , etc ., interleukin - 1 , interleukin - 2 , interleukin - 3 , interleu - ficiency syndrome, which occurs as a result of an HIV kin - 4 , interleukin - 5 , interleukin - 6 , etc . , tumor necrosis fac infection ; and the like . tor, tumor necrosis factor- a , granuloycte colony - stimulating 55 In some embodiments , an siRNA is directed against a factor ( G - CSF ) , granulocyte -macrophage colony - stimulat - member of a signal transduction pathway , e . g ., the insulin ing factor (GM - CSF ), macrophage colony -stimulating fac pathway, including AKT1 - 3 , CBL , CBLB , EIF4EBP1 , tor , midkine (MD ) , and thymopoietin . Still other peptidyl FOXO1A , FOXO3A , FRAP1 , GSK3A , GSK3B , IGF1 , drugs that can be advantageously delivered using the meth - IGFIR , INPP5D , INSR , IRS1 , MLLT7 , PDPK1 , PIK3CA , odology and formulations of the present invention include 60 PIK3CB , PIK3R1, PIK3R2 , PPP2R2B , PTEN , RPS6 , endorphins ( e . g ., dermorphin , dynorphin , a - endorphin , RPS6KA1, RPX6KA3 , SGK , TSC1, TSC2, and XPO1) ; an B - endorphin , y -endorphin , sigma- endorphin , [ Leulenkeph - apoptotic pathway (CASP3 , 6 ,7 , 8 , 9 , DSH1 /2 , P110 , P85 , alin , [Met Jenkephalin , substance P ), kinins (e .g . , bradyki PDK1/ 2 , CATENIN , HSP90 , CDC37 , P23 , BAD , BCLXL , nin , potentiator B , bradykinin potentiator C , kallidin ), BCL2, SMAC , and others ); and pathways involved in DNA LHRH analogues ( e . g ., buserelin , deslorelin , fertirelin , gos - 65 damage , cell cycle , and the like (p53 , MDM2, CHK1/ 2 , erelin , histrelin , leuprolide, lutrelin , nafarelin , tryptorelin ), BRCA1/ 2 , ATM , ATR , P15INK4 , P27 , P21, SKP2 , and the coagulation factors , such as Q ,- antitrypsin , Az- mac - CDC25C / A , 14 -3 - 3, PLK , RB , CDK4 , GLUT4 , Inos , Mtor, US 10 ,000 , 582 B2 23 24 FKBP, PPAR , RXR , ER ). Similarly , genes involved in The sulfur content of the ES -HyA was evaluated to immune system function including TNFR1, IL - IR , IRAK1/ estimate the degree of sulfonation that was required to 2 , TRAF2 , TRAF6 , TRADD , FADD , IKK? , IKKy, IKKB, achieve this improved glycolytic resistance , and it was IKKO , IkBa , IkBB , p50 , p65, Rac , RhoA , Cdc42, ROCK , estimated that the substation ratio was approximately 10 % Pak1 /2 / 3 / 4 / 5 /6 , IAP, HDAC1 /2 , CBP , B - TrCP, Rip2 / 4 , and 5 (FIG . 2 ). This would indicate that for every 10 disaccharide others are also important targets for siRNAs , where such HyA units, there was one conjugated ethylsulfonate . The siRNAs can be useful in treating immune system disorders . other 90 % of HyA units are unmodified and able to partici siRNAs specific for gene products involved in apoptosis , pate in conjugation reactions with other functionalized mol such as Dsh1/ 2 , PTEN , P110 (pan ), P85 , PDK1/ 2 , Akt1, ecules. These results were verified by conjugating HyA and Akt2 , Akt (pan ) , p7056K , GSK3B , PP2A ( cat ), ß - catenin , 10 ES -HyA with EMCH , a heterobifunctional crosslinker that HSP90 , Cdc37 /p50 , P23 , Bad , BclxL , Bc12 , Smac / Diablo , reacts with the carboxylic acid group on the HyA disaccha and Ask1 are useful in the treatment of diseases that involve ride , at various stoichiometric ratios ( FIG . 3 ) . It was found defects in programmed cell death ( e . g . in the treatment of that the sulfonation of ES -HyA had little effect on the cancer ) . siRNA agents directed against p53 , MDM2, Chk1 / conjugation reaction , although there is evidence that the 2 , BRCA1/ 2 , ATM , ATR , p15 /NK4 , P27 , P21 , Skp2 , Cdc25C / 15 available binding sites per HyA molecule may saturate at A , 14 - 3 -3sigma / ? , PLK , Rb , Cdk4 , Glut4 , iNOS, mTOR , lower conjugation ratios, which is likely due to the previous FKBP , PPARy , RXRa , ERa , and related genes can be used conjugation reaction with aminoethylsulfonate . to treat diseases associated with disruptions in DNA repair , These results indicate that the method of sulfonation and cell cycle abnormalities, where such diseases include could dramatically improve the utility of HyA in medical cancer. Examples of such siRNAs and targets are known in 20 technologies. Previous methods of stabilizing HyA using the art; see , e . g ., US Patent Publication No . 2005 /0246794 . sulfation or sulfonation require high -substitution ratios ( 2 - 4 For example , a recombinant retroviral vector that includes a sulfates per Hy , disaccharide unit ) and the reactions must heterologous nucleic acid encoding an siRNA is useful for be performed in organic solvents . By contrast, the method treating disorders resulting from or associated with dysregu - described above can be carried out in aqueous solutions ; lated cell cycle , e . g . , cancer. 25 thus the ES -Hy A products can be more readily translated into biocompatible products . It has been demonstrated that EXAMPLES the stability of ES -HyA is achieved with dramatically fewer sulfonate groups per HyA disaccharide unit . Thus, the ES The following examples are put forth so as to provide HyA can be further modified and used as a substrate for a those of ordinary skill in the art with a complete disclosure 30 variety of medical technologies. and description of how to make and use the present inven tion , and are not intended to limit the scope of what the Example 2 : Synthesis of Sulfated Hyaluronic Acid inventors regard as their invention nor are they intended to represent that the experiments below are all or the only Sulfated hyaluronic acid was synthesized by a two - step experiments performed . Efforts have been made to ensure 35 process . First , aldehyde derivative of hyaluronic acid accuracy with respect to numbers used ( e . g . amounts , tem - (HyAALD ) was synthesized by sodium periodate oxidation perature , etc . ) but some experimental errors and deviations of hyaluronic acid (HyA ) . HyA of 1 million Daltons was should be accounted for . Unless indicated otherwise , parts incubated with sodium periodate (NalO2 ) in the dark at are parts by weight, molecular weight is weight average room temperature to convert the vicinal diols to adjacent molecular weight, temperature is in degrees Celsius, and 40 aldehydes to produce HyAALD (FIG . 4A ) . Second , sulfated pressure is at or near atmospheric . Standard abbreviations hyaluronic acid (SHYA ) was synthesized by conjugation of may be used , e . g . , bp , base pair ( s ) ; kb , kilobase ( s ) ; pl, 2 - aminoethyl hydrogen sulfate to HyAALD and subsequent picoliter ( s ) ; s or sec , second ( s ) ; min , minute ( s ); h or hr, reduction using sodium cynoborohydride. HyAALD pro hour ( s ) ; aa, amino acid ( s ) ; kb , kilobase ( s ) ; bp , base duced in the first reaction step was combined with 2 - amino pair ( s ) ; nt, nucleotide ( s ) ; i . m . , intramuscular ( ly ) ; i. p . , intra - 45 ethyl hydrogen sulfate (NHCH2CH2 - S04 ) and the reac peritoneal ( ly ) ; s . c ., subcutaneous ( ly ); and the like . tion product was incubated with sodium cynoborohydride ( NaBH CN ) at room temperature to produce SHYA (FIG . Example 1 : Sulfonation of Hyaluronic Acid 4B ) . Validation of sulfation of hyaluronic acid was performed ES -HyA was synthesized using via conjugation of the 50 by NMR analysis . Proton -( ' H ) -NMR spectra were produced aminoethylsulfonate to the HyA backbone . The ability of and compared for HYAALD and SHYA to demonstrate the ES -HyA to resist enzymatic degradation relative to HyA was disappearance of aldehyde groups following reaction of evaluated by incubating ES -HyA and Hy , with hyaluroni- HyAALD with 2 - aminoethyl hydrogen sulfate (FIG . 5A ). dase at various concentrations for 2 . 5 hours (FIG . 1 ) . The FIG . 5B , a zoom - in of the area boxed in FIG . 5A , clearly cumulative molecular weight distribution was evaluated at 55 indicated a lack of aldehyde peaks (CHO ) in SHYA as various time points during the experimental interval using compared to HYAADL . size exclusion chromatography with multi - angle light scat - Further validation of sulfation of hyaluronic acid was tering analysis (SEC -MALS ) . It was found that the ES - HyA performed by 2D Proton ( ' H ) - Carbon (13C ) NMR analysis . was able to resist the activity of the glycolytic enzyme, 2D Proton ( H ) -Carbon (13C ) NMR spectra were produced exhibited by molecular weights that were 2 - 3 times greater 60 and compared for HYAALD and SHYA to demonstrate the than the HyA at physiologically relavant concentrations of presence of sulfate groups (SO2 ) on sulfated hyaluronic hyaluronidase ( 0 . 25 - 2 . 5 units/ mL ) . At these concentrations, acid ( SHYA ) (FIG . 6 ) . the half - life of ES -HyA appeared to be in the range of Functional validation of sulfation of hyaluronic acid was 6 . 6 - 26 . 9 hrs , whereas the half- life of HyA was in the range performed by platelet adhesion assay . Platelet rich plasma of 2 . 35 - 7 . 5 . Thus the method of sulfonation increased the 65 (PRP ) was collected from blood and used for platelet half - life of the biopolymer by approximately 3x on a con - adhesion experiment performed on heparin , native centration controlled basis . hyaluronic acid (HyA ) , and sulfated hyaluronic acid (SHYA ) US 10 , 000 , 582 B2 25 26 with varied degrees of sulfation . Degree of sulfation was tive cells were observed indicating in situ differentiation of varied by varying the moles of 2 - aminoethyl hydrogen CPCs to endothelial cells and vascular morphology became sulfate reacted with HyAALD (25 , 50 , or 100 moles of apparent after 12 days in culture (FIG . 12 ) . 2 -aminoethyl hydrogen sulfate ). Platelet adhesion experi While the present invention has been described with ments were visualized by scanning electron microscope 5 reference to the specific embodiments thereof, it should be (SEM ) imaging ( FIG . 7 ) . The experiment demonstrated understood by those skilled in the art that various changes platelet aggregation on native HyA but no aggregation of may be made and equivalents may be substituted without platelets on SHYA or heparin control. departing from the true spirit and scope of the invention . In Further functional validation of sulfation of hyaluronic addition , many modifications may be made to adapt a acid was performed by clotting assay on platelet poor 10 particular situation , material, composition of matter, pro plasma. Platelet poor plasma (PPP ) was collected from cess , process step or steps, to the objective, spirit and scope blood and recalcified in the presence of sulfated hyaluronic of the present invention . All such modifications are intended acid (SHYA ) with varied degrees of sulfation . Degree of to be within the scope of the claims appended hereto . sulfation was varied by varying the moles of 2 - aminoethyl What is claimed is : hydrogen sulfate reacted with HyAALD (25 , 50 , or 100 15 1 . A sulfonated hyaluronic acid matrix comprising a moles of 2 - aminoethyl hydrogen sulfate ) . The modulus of sulfonated hyaluronic acid polymer comprising a sulfonate clot formed by recalcification of PPP was analyzed on containing moiety coupled to a reactive aldehyde group of oscillatory rheometer and the results were plotted (FIG . 8 ). an oxidized hyaluronic acid , wherein the sulfonate -contain The experiment demonstrated that the modulus of clotted ing moiety is selected from the group consisting of: 3 - oxo PPP decreased with the increase of degree of sulfation as 20 propane - 1 -sulfonate , 2 - aminoethanesulfonate, and 2 -amino higher sulfation inhibits the recalcification of PPP . ethyl hydrogen sulfate . Further functional validation of sulfation of hyaluronic 2 . An implantable sulfonated hyaluronic acid material acid was performed by carbazole assay . Enzymatic degra - comprising : dation of HyA and sulfated hyaluronic acid (SHYA ) with a ) a sulfonated hyaluronic acid matrix according to claim varied degrees of sulfation was measured by carbazole assay 25 1 ; and in 15 U /mL of HAase solution at pH 7 . 4 . Degree of sulfation b ) a living cell and / or an active agent. was varied by varying the moles of 2 -aminoethyl hydrogen 3 . The implantable sulfonated hyaluronic acid material of sulfate reacted with HyAALD (25 , 50 , or 100 moles of claim 2 , wherein the living cell is a stem cell , a neural cell , 2 -aminoethyl hydrogen sulfate ) . Cumulative degradation a neural progenitor cell , a muscle cell , a muscle progenitor was plotted over time (FIG . 9 ) . The experiment demon - 30 cell , a skin cell , an endothelial cell, or an epithelial cell. strated that the enzymatic stability of HyA increased with 4 . A sulfonated hyaluronic acid drug delivery composition increasing degrees of sulfation . comprising : Further functional validation of sulfation of hyaluronic a ) a sulfonated hyaluronic acid matrix according to claim acid was performed by retention kinetics assay . The kinetics 1 ; and of retention of TGFB1 was measured in sulfated hyaluronic 35 b ) an active agent associated with , embedded with , or acid (SHYA ) with varied degrees of sulfation . Degree of encapsulated within , the sulfonated hyaluronic acid sulfation was varied by varying the moles of 2 - aminoethyl matrix hydrogen sulfate reacted with HyAALD ( 25 , 50 , or 100 5 . The sulfonated hyaluronic acid matrix of claim 1 , moles of 2 -aminoethyl hydrogen sulfate ) . The percentage of wherein the sulfonated hyaluronic acid polymer has a sul 40 nM TGFB1 retained by the differently sulfated hydrogels 40 fonate : disaccharide ratio of 1 or less . was determined by ELISA and plotted over time (FIG . 10 ). 6 . The sulfonated hyaluronic acid matrix of claim 1, The experiment demonstrated that retention of TGFB1 is wherein the sulfonated hyaluronic acid polymer has a sul dependent on the degree of sulfation of the SHYA with the fonate :disaccharide ratio of 0 . 5 or less . most sulfated HyA hydrogels ( 100SHYA ) retaining over 7 . The sulfonated hyaluronic acid matrix of claim 1 , 70 % of the TGFB1 for up to 20 days . 45 wherein the sulfonated hyaluronic acid polymer has a sul fonate: disaccharide ratio of 0 . 1 or less . Example 3 : Effects of HyA Sulfation on Cells 8 . The sulfonated hyaluronic acid matrix of claim 1 , wherein the sulfonated hyaluronic acid polymer has an in The viability , proliferation , and adhesion of cardiac pro - vivo half - life of 8 hours or more . genitor cells (CPC ) was assessed in sulfated hyaluronic acid 50 9 . The sulfonated hyaluronic acid matrix of claim 1 , ( SHYA ) with varied degrees of sulfation . Degree of sulfation wherein the sulfonated hyaluronic acid polymer has an in was varied by varying the moles of 2 -aminoethyl hydrogen vivo half - life of 10 hours or more . sulfate reacted with HyAALD (25 , 50 , or 100 moles of 10 . The sulfonated hyaluronic acid matrix of claim 1 , 2 - aminoethyl hydrogen sulfate ) . Cells encapsulated in sul- wherein the sulfonated hyaluronic acid polymer has an in fated HyA hydrogels demonstrated high viability after one 55 vivo half - life of 12 hours or more . day in culture, as assessed by double staining with calcein 11. The sulfonated hyaluronic acid matrix of claim 1 , ( green , live cells ) and propidium iodide (red , dead cells ) wherein the sulfonated hyaluronic acid polymer has a ( FIG . 11 , top panels ) . CPCs were capable of adhering and molecular weight of 100 kDa or more . spreading within the sulfated hydrogel networks containing 12 . The sulfonated hyaluronic acid matrix of claim 1 , the adhesive ligand bspRGD ( 15 ) , as assessed by double 60 wherein the sulfonated hyaluronic acid polymer has a staining for f -actin stress fibers ( TRITC -phalloidin , red ) and molecular weight of 400 kDa or more . nuclei (DAPI , blue ) ( FIG . 11 , bottom panels ) . 13. The sulfonated hyaluronic acid matrix of claim 1 , CPC differentiation was also assessed in sulfated wherein the sulfonated hyaluronic acid polymer has a hyaluronic acid ( SHYA ) with varied degrees of sulfation . molecular weight of 800 kDa or more . CPCs encapsulated in sulfated hydrogels containing 65 14 . The sulfonated hyaluronic acid matrix of claim 1 , bspRGD ( 15 ) and TGFB1 were stained for the endothelial comprising one or more additional moieties selected from a cellmarker CD31 using immunohistochemistry . CD31 posi crosslinker, a polypeptide, a label , and a drug . US 10 ,000 ,582 B2 27 28 15 . The sulfonated hyaluronic acid matrix of claim 1 , comprising a polypeptide conjugated to the sulfonated hyaluronic acid polymer . 16 . The sulfonated hyaluronic acid matrix of claim 15 , wherein the polypeptide has a molecular weight of from 1 5 kDa to about 2000 kDa. 17 . The sulfonated hyaluronic acid matrix of claim 1 , wherein the oxidized hyaluronic acid comprises a D -glu curonic acid ring that is cleaved to form two reactive aldehyde groups . 10 * * * *