US 2011 O189287A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0189287 A1 Abbott et al. (43) Pub. Date: Aug. 4, 2011
(54) METHODS AND COMPOSITIONS FOR Publication Classification WOUND HEALING (51) Int. Cl. A6IR 9/14 (2006.01) (76) Inventors: Nicholas L. Abbott, Madison, WI A6IR 9/00 (2006.01) (US); Ankit Agarwal, Madison, WI A63L/78 (2006.01) (US); Christopher J. Murphy, A6II 35/12 (2006.01) Davis, CA (US); Jonathan F. A6IP 7/02 (2006.01) McAnulty, Oregon, WI (US) CI2N 5/00 (2006.01) B82Y5/00 (2011.01) (52) U.S. Cl...... 424/484; 424/400; 424/78.31; (21) Appl. No.: 13/024,856 424/93.7; 435/325;977/755 (22) Filed: Feb. 10, 2011 (57) ABSTRACT The present invention relates to methods and compositions Related U.S. Application Data for wound healing. In particular, the present invention relates to promoting and enhancing wound healing by utilizing (63) Continuation-in-part of application No. 12/363,044, cross-linker covalent modification molecules to attach and filed on Jan. 30, 2009. deliver wound active agents to a wound. In addition, the present invention provides methods and compositions utiliz ing oppositely charged polyelectrolytes to form a polyelec (60) Provisional application No. 61/303,104, filed on Feb. trolyte layer on a wound surface. The invention further relates 10, 2010, provisional application No. 61/024,725, to incorporating wound active agents into a polyelectrolyte filed on Jan. 30, 2008. layer for delivery to a wound. OO Y.
Patent Application Publication Aug. 4, 2011 Sheet 1 of 28 US 2011/O1892.87 A1
FGURE 1
Patent Application Publication Aug. 4, 2011 Sheet 2 of 28 US 2011/O1892.87 A1
FGURE 2 200 N. -240. X X x X X k l l 230 l X
220 210 Patent Application Publication US 2011/O1892.87 A1
Patent Application Publication Aug. 4, 2011 Sheet 4 of 28 US 2011/O1892.87 A1
FGURE 4
Polyelectrolytes wound bed, 30 minutes cycles
: PBS-PSS x: PBS-PAH : PSS--PA Patent Application Publication Aug. 4, 2011 Sheet 5 of 28 US 2011/O1892.87 A1
FIGURES
Wildtype vs. Db/Db Mouse Controls - Wound Area Day 9 Post Wounding
an C E S. g x Db/Db s Widtype o f s C
Day 9 Post Wounding Patent Application Publication Aug. 4, 2011 Sheet 6 of 28 US 2011/01892.87 A1
FIGURE 6
Activation with Activated beads
NHS/EDC
Growth factors 8 8 9. Patent Application Publication Aug. 4, 2011 Sheet 7 of 28 US 2011/O1892.87 A1
FGURE 7A EE): AAP EOMMA DEMANIENSAE) EXE: ANDROPN RECRSOR BE: ANXN ANMCREA PRERSC) NSEUEFENCEEE, EEET). : AFP CAS ID: ANDP. BROOR {}): A BAE APIME O:A.N.PNGA RCEN E: ANON PRERSOSR. iDE: ABAECN PREC:RSOR, NSEC: NSEC BEENCE: PEEE DEENCE PERE II); ALOACRLC) E: ANOROBA PEPXE ID: ANEP ROSE }}): ABAE EOMPA AO). DE: ANROFN PREC:RSOE, EE ABAECN INSEC: EPESCE, NSECEFENE PEPE EBE, ii); ALO2 ACRLO EANVECREBALEE ED AND ORS D: ABAS BASA AEO-. EE: ANDROPN PRECURSOR BE: APA-EASRRE3RN NSEC BEENCEEE (FRAGMENI). OAO ACRLX DE ANMROBAEEEEEEE X: A RE ID: ABF CAEEL AO DE: ANDROPIN PRECURSOR, DE: ABF-. ANEBACEERA NSE BEENCE EE ACOR PREC:RSOR: NEwAEO33E ): AS MO SN, INSECE DEF : AN-POYSACCHARDE EAN RYA (LPS) FACTOR, DE: ANRON PRECRSOER NSEC DEFENCE PEPE ED: ABF2CAEEL II); ALPS TACTR BE; AB-2, ANTIBACKERAL EAN-IPOPYSACCARE ED: ANID, ANDA Ac). PRE EACE (LPS) FACTOR. BE: ANCRON: CEECERAE BEFENCE PEOE ID, AMP, ALLCE DE ANMCRB.A. ROTER DARP ANOSA. II: ABF5. CAEEL ACE-AMP PRECRSOR PLAN E: ARNPN D: ABF-3 ANBACERA EFENCE PEPE FACTOR PRECURSOR: NE&AEO{}}E ID: ANSA SIRCZ EFENCE PEPE II); AMPE MACEN OE ANEE3ABEA SBSEANCE OE: AMEROBAL PEPDE A. By: ABF CAEEL. PRECURS OR (AMP, PLANT BE; ABF-8, ANTIBACKERAL DEFENCE PEPTIDE; ID: ANTFSARPE FACER PRERSORNEAODE ANTN.GAL PRON BEFENCE FEDE IDAMP). MESCR 'RSOR (AFP). ENSEC: DE ANCREBAPEEEEEE ENCE EXE II); ABP2 RIPCL PRECFRSOR ANTEFENCE ED: APE4 APIME OEANIBACERAL PEPTOE PEPEEDE E: ARIAECIN PRECJRSCR, YPE PRECURSOR PUTATIVE). INSECT 4. INSECTDEFENCEPEPTE ESENCE PEPE IE). AMPE MIRA OE:ANIMCROBAL PEPTE IE): AP22 APIME B: ABPP RIPCL (AMPI): PLANTEEFENCE FEFFIDE DE: APIDAECEN FRECFRSOR, IYPE E: ANTERACERA, PEPTOE 22 NSEE EFENCE PEPE PRECORSOR (PUTATIVE). INSECT ID: AMP2 MIRA EEFENCE PEPOE DE: ANMCROBAL PEPTE 2 ID: AP39 PIG (AMP2), PLANT DEFENCE PEPFIDE BEAN3AEREAL PEPE {E}: ACA ACALU B: AMP AMACA 398. MAA ANDEFENCE EBE: ACACEEERIN A INSECE EAN3AEEEDE EDE DEFENCE PEFFIDE: COLEPIERICIN (AMP1 AND AMP2), PIANT EFENCE PEPIDE. ID: AP73 APIME II: ACANACAGO E: NSECT ANIMICROBIAL EAANOSRRN ?E). AMPA MAHY PEPE PRECRSR E: ANMCRORAL PROE RECFRSOR PAN DEFENSE Do: APA2 BOVIN }}): ACHCACHFU EEE DE: APOLIPOPROTEIN A-II (APO DE: ACHACN FRECRSOR: AII). ANTIMICROBIAL PEPTIDE GASTROPODEFENCE PROEN. ID: A.M.P. CARMA BAMP-1), E: CARCINN, KA EE). AFP2 GASEL AMICROBAI, FRIEN: ED: API2 APIME BE: ANNGA ROEN. RSEACEAN EFENSE PROEN DE: AAECN NSET DEFENCE EIDE {{): AFPs. BRANA ID: AMSIE BOVIN E: CYSTEN-RECHANTIFNGAL OEMEANROPNAPHA ID: APIA APIME PIEPTIDE FREC FRSOR; PIANT (AIP; A-MSH) D: PRO-AAECN A. ENSRC EENSN EEENE PEPEE
ID: ANOP DROME ID: APIB APIME }}}: AFPASPG| Patent Application Publication Aug. 4, 2011 Sheet 8 of 28 US 2011/O1892.87 A1
FIGURE 7B E: ER-APAECNE3. NESEC DE: GAILNACN AIFHA, AVLAN DEFENCE EDE }}): BAEA MAIZE BEA-EFENSN DE 8ASA. AER ANIFNA !): API BOMPA PEPSDE PRECURSOR, i}: 8)01B PRECB E: APDAECIN.INSECT). FENCE E: DEFB-LKE PROEN. REFEEE ii): BAP3A MAIZE E. BASA AYERANNGA II: BioP MOUSE ID: AFI.Y AFLKLF FEERERSR E: PRSAE BEA-EFENSN E, AYSLANN-A RECORSCB& GASROBEEENCEPROEEEN. ED: BAP3B MAEZE D: B AFPA E. BASA AYER ANCSA E. SPHENISCEN E SPE-) (PBS)- ED. APRFB BOVEN FEDERERS 1}(AVIAN BEIA-DEFENSIN) BEAPROTENNANEBACERA ERAGMENTS ED: BCT PRC BOVIN {{): BD01, BOVEN D: CYCLECOCOECAEPDE EBCWNEBETA-DEFENSIN 1. HD: ARINCICAR PRECIRSOR (BNEDE3-1). EDE: ARIETIN FRAGMENT); PLANT BCE). CATHELICED-IN) DEFENCEEPE. ID: BCT PRC SHEEP BD: BDO CAFIII DECYCCOEAPEPE DE: BEADEFENSIN FRECURSOR ID: ASABF ASCSU PRECRSOR (BE3-E. E:ASAACERA. ACOR (BCT (CATEEELICEDEN). PRECRSORNEMATOE j}: BD01 CERAE EFENSIN NSECE EENSIN }}): Boii BOVIN DE: CERCOPECUSAEIOPS KE E. CYCAPEPE). CERCOPTHECS BETA-DEFENSN CAEB)-. II: ASCP2 ASCTR ID: BCT SHEEP EDE: ASCAPHIN-1, -2. AN3) 3. FROG OE. SHEER CYCC {{D: BIO) CERER DEFENCE PERTIES (ECAPEPTE E: EROFHECUS ERYHROASTER BETA ASCP, ASR ID: BCISPR BOVIN {DEFENSIN E (CERB (-). EDE: ASCAF'HN-4, -5,-6 AND 7. EDE: BACTENE CEN 5 (BACS) EROG DEEENCEFEEIDES FRECURSORCATHE CEDEN), ED: BOO1 CERPR E: ERCOFIHECFS PRESS ED:ASCPS ASCIR EE). BCT51R CAPHE BETA-DEFENSIN (CPRBE)-1), EE: ASCAPHIN-S, FROG DEFENCE OE BACENECENS RECORSOR PEPIDES (CHBAC5)(CATHELICHON). BD: BDO CHICK DE: GANACN AWAN BETA ID: ATT DROME DEFENSIN. EAACN BE PRECRSO3. }}}: 8CISPRC SHEE? E. BACENECN 5PRECJRSR }}}: 80E CHILA }}}: ATTA DROME (BA5)(CAIHELC}}}EN) EAAN ARERS }}: 8D01, DOG }}}: 8cis BOVIN E: EEEA.DEFENSIN. KEPEPEEE IT): ATA GTOMO E: EACERNECIN 5, FACS. | CANTS E ANWR3A, EPE ATACIN ATFA, INSECT DEFENCE, ID: BCT6PRC SHEEP : B0} EQUCA PROTEN E BAC.NECN 6PRECRSOR OE: HORSE BEA-DEFENSIN (CAFIHELICEDIN) ID: ATA TRINE {{): BIOG) GORGO DE: AACN ARECRSOR ID: BCT 7PRC BOVIN E: CORIA ORLABETA NSECE DEFENCEFROTEIN EDE: BACTENECIN 7 (BAC7) OEFENSIN (CGCBD-ly. PRECURSCR (CATHELICIDIN). HD: ATTB HYACE {E). BOO1 HUMAN s: PRARACINEORS BE: MAN BEA-EFENSIN (IMMUNE PROTEEN P5); INSECF : 8 FPRC SEEEE: (HBE)-i}. EFENCE PRON BACTENECIN 7.5 (BAC7.5) PRECURSOR (CATHELECDIN). is): BE30: HYLCO HD: A FE HYACE }}E: ATTACIN E ANDF (MMJNE ED: BCT BOVINOE: BA("ENECIN B. : BEXO HYLA PROTEIN P5), NSFC DEFENCE 7, BAC7. DE: HYOBATES ARBEA PRCENS BEFENSIN }. (H). ARBE)-E). }}): BCT CARMADE: : A BOMMC IRSACEAN EACENECN
EAAN PRECRSORNSEC (FRAGMEN) ID: BDO HYLMO DEFENCE PROEN DE: YOBAESMOOCHEBEA ED: BCT7 SEIEEPIDE: SHEEE BEFENSIN (EEMOLBD-1. ID: BABIB MAIZE E3ACTENECNEAC.S. RE: BASA.AYERANTIFINGAI REFEDERRECRSOR, }}}: 83D0EACHICK {{): BI-01 MACFA
Patent Application Publication Aug. 4, 2011 Sheet 10 of 28 US 2011/O1892.87 A1
FIGURE 7D JE): BLP BOMOR ED: BOME IS BOMVA E BOMEBNN.KE PERE DE BOMBININ HS FRECURSOR (BLP-1), II: BIO(36 BOVIN t): BOMN BOMVA E: BOWINE BEA-BEFENSN 6 i}): BLP2 BOMOR OE: 30MBININ. (BNEDB-6). DE: BOMBININ-KEEE 2 (B} P-2). ID: BP BOVIN ID: BD06. MOSE DE: BENE EACERCA E: BEA-XEENSN 6. PERMEABLIY-NCREASENG IE: BLP3 BOMOR FROTEIN(BFI). T}): Bä)07 BOVIN DE: WBNN-RE, PEPER 3 DE 8OWNEBEA-DEFENSIN 7 PRECURSOR (BLP-3). ID: BPI HUMAN (BNDB-7). DE: 3MAN EBACERA IED: BLP4 BOMOR PERMEABLIY INCREASNG IE: BE)07 MOUSE E BMBININ-KEEPE 4 PROTEIN PREC, FRSOR (BPI) E BEA-DEFENSN (BSP-4, (CAP57). RECRSs. (E). BLP BOMVA ID: BPI RABIF BE BOBININKE PEPE DE: RABBTBACTERICAL TO: BO08 BOVTN PRECLRSOR (BLP). FERMEABILIY-NCRESN DE BOVINEBEA-DEFENSIN 8 PROTEN (BPE) (FRAGMENT). (BNDB-8). IED: BMAF27PRC BOVIN BE: ANEBA(ERAL PERIOE }}}: BPI BOVIN TO: Boo8 MOUSE BMAP-27 PRECURSOR: DE: RACERCOA FRAGMENTS E: BEIA-DEFENSIN 8 (BEEA CAHELICONME327 IN FROM BPTI. APROTENIN DEFENSIN 6). SWSSRC ID: BRA RANES ID: BD09 BOVIN IE). BMAF28PRCBOVIN DE: BREWENN-EA DE: BOWNE BEA-DEFENSIN 9 9 ANACRAPP) (BNDE-9). BMAP-28 PRECURSOR: ED: BREA RANTE CAELICON. 28 N E: BREWENN.A ID: BDO BOVIN SWISSPROT E: E3ONE BEA-DEFENSIN 0 ii); BRIBA RANBE (BNDB-10). IED: BMAP34 BOVIN DE: BREVENNBA DES ANTBACERIA PEPIDE IE): BE) 18OVIN BMAP-34 PRECURSOR, ME334. FE). BR BB RANBP E. BOWNEBEADEFENSIN DE: BREVENN-IBB. (BNDE3- ). {E). BMKBPP. BUTMA DE: BMKBPI (PARABGEOIORIN). ID: BRIBC RANDE OF BREWNN-BC. IE): BE) 12 BOVIN IE): BNP32. HUMAN BE: BOWNEBEADEFENSIN 2 E. BRAN NARREEC PEPERE (BNDE3-12). (BNE-32). }}}: BRIBD RANBE E: BREWNN.B. JE: BE) i3 BOVIN ID: BOL1 MEGPE BE: BOWNEBEA-BEFENSIN3 DE: BWBOTN. ED: BRiBE RANBE (BNEB i3). DE BREWENN.B.E. it). BOT 2 MEGPE I}): B40 MOUSE BE BOMBOLIN. ID: BRB RAN BE E. MSC:SE BEAEFENSN: DE: BEREVENN-BF. PRERSR IE): BOL3 MEPE DE: HOWBONE : BRBYA RANBO ID: BDC7 B.VIN DE: BREWNN-BYA-BYBANO - BE: BEA-E ENSNCF EE). BOL. MEGPE BYC. PRECURSOR; BIBIO-C7 E BOMBOINV (EFRAGMENT). ED BREBRANES ii); BOL5 MEGPE DE: BEWNN-EB. ID: BHP BOVEN: E: OMBIN. W. E: EBOWNAPAEEMOGLOBIN ANSIMICROBAL FRAGMENT O: BOMH BOMEA FROM CK) DE BRBNIN ED. FR FC RANFS E. BREWNN-EC. IB: BCDEF LIMPO IE): EOME 2 BOMVA E. LIVES BIG EFENSEN BF: OyNIN | D: BRE, RANES l{}: BENE RAE i}). BOMH3 BOMVA DE BREWINNE PRECRSR. XE ANMICRBIALIKE RE: BOMBININ3. PROTEIN BEN-1B RATUS ID: BREA RANL} NORVEGECOS. IE): BOME34 BOMVA DE: BREWENN-LA BE: BOMBNN 4. ED: BRB B RANL{} Patent Application Publication Aug. 4, 2011 Sheet 11 of 28 US 2011/O1892.87 A1
FIGURE 7) E BREWINN-IB IDBRTB RANTE EAROCN CAN E: EWNN-28;&SCR. ANTI MICROBAL PROTEIN CAP37) if). BRPHRANP (HEPARIN-BINDING PROTEIN) BE: BREWNN-PB, ID: BUTILANIDAF (HBP). DE: BJEHINEN CENELECERATE HD: BRSC RANSP DEFENCE EPLE ii): CASN1 BOVEN XE:BREWINNES OE: CASOCON-APHA-S2 II. C.8FRC RABE} CASEIN FRAGMENT (65-203). BD: E3R.S. RANSP E: 8KOPOSACCARE EEREWNN SAAN SE3 BENDING PROSEIN PRECURSOR(18 {{): 'A'E (; H MAN KD (ATIONEC PROTEIN) (CAP18-A) {}F: 'A' HEPSIN GPRECURSOR (EC : R A RANA (CATHELICIDEN). 3.4.2.2). OE: BREWINN.TRA ID: C8 RABIT ED: CATH29 MYXGL. ID: BRT RANTE 3: CAP8 E EMAPOEI BE: BREWNN ANVR3A, PE-29 ID: CAEE XENLA PRECRSOR II); BR. RANBP E: PRECAE&LENYPE (CAEI29)(EIFIAP}(FRAGMENT)(CAF DE BREWNN (ANTIMICROBIA CPF HELICE IN PRECIRSOR) s: 8R RANA O: AT-3 MYXG. EBREWNN-A. II: CAE2. XENLA E: E.AOPEC E. REPROCAERENEYE I ANCEBAEE-3 E: ER20A RANOR (FRAGMEN}). ANEEMECROBIA). SECRSR WNEN-2A CPF PRECIRSOR) (CATH37)(HFIAP)(CATHEE ICTDIN) ID: BR.20B RANOR ID: CAES XENEA BD: CATII DOG E: BREWINN-20B, DE: PREPROCAERENYPE II. EOG CATHE CON. (AN' MCROBALC? D: BRA RANES PRECURSOR) CE EA E: BREWNN-IEA ECESOPERSBR. ID: CAE4XENBC IE): BR2B RANES DE PREPROCAERENYPEW ii): CEC1. MUSIDO E: BREWNN-2E.B. (ANTIMICROBIAL CFF E: CFCROPN PRECURSOR) HD: BR2C RANES {D: CECI PIG EEBREWINNEC. JD. CAEA XENE.A. E: ECR3PN. E: REPROAR ENEYE W. BD: BR2D RANES (ANTIMICROBIAL CPF EE). CEC2 CERCA DE: BREWNEN-2EEB. PREC, FRSOR) BE: CECROPN2 PRECRS OR i: BR2 RANES II: CAE5 XEN LA HD: CEC2 MANSE E. BREWNN-2E. DE: PREPROCAERULEIN (CLONE E. BACERCNE-2 PXC202) (FRACMENTy (CECROPIN-LIKE PEPTIDE B-2). ID: BR.2F RANES (ANTIMICROBIAL CPF DE: BREWNN-2EF PRECORSOR PRECORSOR). }}): CEC3 MANSE E. ACERCN-3 E3: BR2G. RANES ID: CALN. ONKW (CECROPIN-LIKE PEPTIOE B-3). EEBREW NEN-2EG, AMPHIBIAN OE: ALLWCIN, DEFENCE FETEDE | D: CEC4 BOMMO II). CALL BOWN E: CECROPIN (ANEBACERA }}}: BR2H RANES J.F. CAI TRINSFMINAL FEERIDE CA-W). DE: EREVENIN-2EEH AMEFEBEAN PLASMIN PRECURSOR DEFENCE EDE FRAGMENT). ED: CEC4. MANSE E BACERCON B-4 i: BR2 RANBP II: CAP7 AH MAN (CECROPIN-LiKE PEPEL)E B-4). E BREWNN-2 E: FRAGMEN 2-24 OF CAP MAN. BD: CEC5 MANSE ID: BREE RANES E BACERCON ESP E: BREWFN-E. PREC RSOR (CECROPIN-EKE, PEPETOE B-5). II); BREE RA NES BE: BREWNN-2EE, ID, CAP: HUMAN li): CECA. AEDAE DE: AZEROCON PRECFRSOR DE: ANTIBOICPEPTOE }}}: BRKM BOMMA (CAI'ONIC ANIMICROBAL. IDE: MAXIMAKININ CONTAINS: PROTEIN CAP37 (HEPARIN D: ECA ORSE BRADYKINEN. BENDING PROTEIN) (BP) BE: ECRRNA RECRSCBR (FRAGMENT). i}): BREARANEE EE). (ECA: HYPCC F: WNEN-A PRCRSOR IT): CAP7 PIG E}E: HYPHANCIN BE PREC RSOR, HYPHANA CECRPNA Patent Application Publication Aug. 4, 2011 Sheet 12 of 28 US 2011/O1892.87 A1
FIGURE 7E DE: AR3C PEPDE D: CECA2 AEDAS CECROPEN C2. l:3: CIRB CHAPA DE: ANICEE DE CIRC LIN B (CIRB). CECROPNA2. II); CECC DROME E: ECROPN RERSOR. II: CLVAPRC STYCL II: CECA2 HYPCL }}E: (T.AVANTN A PREC RSOR: DE: HYPHANCEN IF PREC:RSOR, ID CECD AGRCO NCASE ANMCROBAS PANA ECRON A. BE: CEERINSEE AND2 FEPOE (FRAGMENT), NSECT DEFENCE II); CECA3 FIYPCU PEFES II: C.VA. STYC). TE: HYPHANCEN IG FREC FRSOR, E; LAWANNANICARE YPAN REACEN A3 ID: CECID ANTFE DEFENCE PEPIDE DE CECROPEN. H. CECA AEDAE Ji): C.V STYCL E: CECROPNA I}: CECE). HYACE E: LAWANIN PRECRSCR OE CECRON O PRECRSOR ED: CECA. AEDAL ID: COILE ZOPA: BE: CECROPEN A (AACECA). FD. CFCM BOMMO DE: COLEOPTERICIN INSECT DE: (BOMBYX MORI CECROPIN EFENCEEPE. s: CECA BOM) PRECTERSOR)(CECROPEN). OE: CEROINA PRECRSOR ID: CRI) LITSE ED; CECANOGA DE; CAERN (), II: CECA DROME DE: CECROPN PRECRSOR DE: CECROPINA APRECURSOR, li): CRll LIESP s' CERA CERCA. DE CAERN ID: CECA. HYACE DE CERATOTBXINA DE: CECROPEN A AND C. II: CR 12 LITCE PRECRSOR II: (ERB CERCA E: ERIN 2. E: ERACOXINB, ID' CECA HYPCL ID: CR 13 LITCE OE: YHANCENIE PRECURSOR, ED; CERC CERCA OE, CAERN 3. YPHANTRACECRON A E. CERAOCXNC PRECRS OR II): CR14 LEEGI {L}: CECA TRINI ID: CERE CICAR XE: CAERIN 4, OE CEROINA PRECRSOR EDE: CICERTN (FRAGMENT); PLANT DEFENCE PEPOE, Ji): CR 15 LTCE ECB AEA E AERN S. DE: AN3OICPEPE L}: CG HUMAN CECRON B. {DE: CATHEPSIN G FRAGMENT (230 IB: CRE6 LTXA 255). DE CAERN. H. CECB2 AEDAL. E ANTBOTC PEPTE I: CGA HUMAN E3: (R7 LITXA. CECROPNB2, DE: CAE HEPSING FRAGMENE (S2 DE CAERN 7 101). ED; CECBANEPE II): CR 18 LITCH E: ECROPNB, ED: CGB IIUMAN E AERN 8. DE: (AEHEPSING FRAGMENT (137 II); CECB BOMMO 58). li): CR9 LE: CH OE RINE PRECERS DE CAERN 9. (LEPIEDOPTERANAANI). By (CECH II); CGC HUMAN ANI) CECB2). EE: CATHEPSIN FERAGMEN 20. II: CR2) LIISP 235. E: CAERN2. ID CECB DROMA E CEN PRCFSR ED: CHECATH CHICK T}): CR22. II: GT O AWAN CASECN E. CAERIN 2.2 (CONEANS II); CECBI)ROME CAERBN 2.2.1). E: CECROPINB PRECORSOR ID: CHY PAGMA DE: CHRYSOFESNS AND IID: CR23 LTCE FD. CECB DROSE 2.(9 EUROCIEDIN-LKE PEPTIDE) (DE. SPELENADPHERN CAERIN 2.3). E: ECRON BRECRSXR. 3: CR24. LTCE ED CECB HYACE ID: CHY3 PAGMA DE CAERIN 24. DE CENE PRECS) DE: CRYSOFSN (IMMJNE PROTEIN P9). 3.(pLEUROCIDIN-LERE PEPTIDE) li): "R25 LEEGI II: CECC AEAL E. CAERN 2.5. OEAN BIOTICPEEDE ED CCN CECF). CECRPN C. EE: CICAIN FRAGMENT. ID: CR3. SS DE: CAERN3. II: CECC2 AEEAL II: CIRA CHAPA i)E: CIRCULIN A (CERA). tip: CR.32 LTCF. Patent Application Publication Aug. 4, 2011 Sheet 13 of 28 US 2011/O1892.87 A1
FGURE 7F E: CAERN 33. iD: DBF EEG I: EF ORSE D: CR33 I'ECE DEIAZEPAMINING E. CAERN33. INHIBITOR DBI) FRAGMENT 32 ID: DEFE MYTGA 86. IDE: MYFILUS DEFENSIN (MCE)) HD: CR34 LTCE DE: CAERN34. IE). IDCO HUMAN TD. EDEF RABIT DE: CONANS: C-1}, OE: ID: CR4 LIFCE AN AIRNOCO8 CORON E: CAERN4. ID, DEF is MOFSE PEPHDE 1) E: CYPEN OPRECRSR p: CR42 LYECE (FRAGMEN3), EFENSEN, | D: DEF: RA BE: CAERN 42. E: A NER PEPE : DEF: N8SE (RNP-1 (RTNP-2), RAT E: CRYN-ERSR CORE (COSEAEN RE. R2), (FRAGMENT, DEFENSIN. EFENSN H): CRAMPPRC MOUSE ID: DEF 13 MORSE D: DEF: SAGA E MOSE CAEN REATED E. CRYP-3 PRECRSR E: PRMAE, APEA DEFENSN PROBEINAOSE CATHELINKE (FRAGMENT, DEFENSEN, (FRAGMENT). REN {{): DEF 4 MOOSE C HD: CRAMP MCSE E: YN-4 EECSCR ENSN PRECRSR DE: MCP (MOGSE CATHEN (FRAGMENT, DEFENSIN. SECE DEFENSIN. RELATED PEPFIDE, CRAMP (CATHEELEN RELATED AMP) D: DEF15 MOFSE D. BEF2A MACME} E: CRYPEN SPRECRSR DE: NACACA MUAA AFEA }}: CRAM RA (RAGMEN), El:FENSIN. DEFENSEE-2, READ-2 DE: CRAMP (FRAGMENT), (CAHELIN RELATED AMP). : DEF6 M8SE : BEF2A SOCA CAEEN E: RYBN-6. SSR E BENSN 2A ECSCR (FRAGMENT, DEFENSIN. NSEC BEFENSIN ID: DEF 7 MOSE D: DEF2B MACMt. E. CRYP-PRECRSR DE NACAC WAAAA ID: CRN LITVA (CRYP)7); ALFHA-DEFENSIN 7. IDEFENSEN 2 (NAME CONELIC 3)E: CRSN, K33A W. H. DH:2A MACMI). AMICROBEA PROTEEN: EED: DEFEA STOCA CRFSACEANEEENSEROEEN E: DEFENSNA, RERSOR : EF2 ANCSA INSECTDEFENSN EE N. (i): CRYPMO SI: EE: CRIPTINES. CRY -li, ID: DEF ANOGA LD, EEF2 APIME m NSSEE E: N PRECRSR DE: DEFENSIN (FRAGMENT. }}EEENSINS (3. F NSC33NS DEFx MOUSE). ID: DEF2 MESAU ID: DEF AEEGE E. HASTERNEOPHE E: EAAA ENSN PEPERE2 (-(AN.E.2); NE EROPHI. ID: CTL IITCI (FRAGMENT. DEFENSIN: DE: EXTENDED TO CITROPIN 1..3; Ci'i(OPIN 1.4: D: 1) EF BOMIG ID: DEF2 MO SE. E NSEC EEENSN. IDE: CRYPTION 2 PRECORSOR, O C2 I OEFENSN. DE: 2,3}. D: DEF CENLI. E: SCORNEEENSN II: EEF2 MYTGA ED: CT2 LCI RECRSCBR ESFSEFENSN2 EXE: 2.E. PRECURSOE (MGI2) LD, EEF2 RABIT ; E ERWA DE: }}}: CIA. E. MAN E: BERMACENOR 33-ENSE (ANARENCC(RCOROFIN DE: CALCITERMIN (FRAGMENT OF (PREC RSOR: VARISIN: INSECT PEFFIDE II). CALGRANULIN C. DEFENSIN II): E}}}2 RAE H): CIFA COPSA ID: DEFE MACMSU ERA NER ANBCC E: CJENNNA DE: NEUROPHILDEFENSINS 3 PEPELE NP-2 PRECURSOR: AND 8 PRECURSOR (RMAD). DEFENSN II: CYLAPSYLO DE: CYCLOPSYCHOEREEEA (CEI). ID: DEF1. MESA; I: EF2 SOCA DE: HASTERNE RH DE: DEFENSIN 2 ERECTERSCR EI): D382)O1 A EP: ): . (IANP-1), NE EROPIII, (INSEC F DEFENSINE). 3E OLABELLANN 32. OEFENSIN: Patent Application Publication Aug. 4, 2011 Sheet 14 of 28 US 2011/O1892.87 A1
FGURE 7 IE: DEF3 MACMi F H MAN E: NEEEROPHEELEEFENSINS E3 SN 6. PR32CURSRE)6. II}: DEFE MO FS ANDS PRECURSOR (RMAID). E: ESS DEFENSN RECERSOR ID: DEF6 MOSE (FRAGMENT). IE: DEF3 MESAU OE. CRN-62 PRECRSCBR E: HASTERNETROPHL DEFENSIN. In: DEFR MYFT) PEPTEDE3 (HANP-3):NETROPHIL E: DEFENSN DEFENSN I: DEF6 RABET DE. (MICROBICIDA, PEPFIDE NP ID: DEFBORNMO ID: DEF3 MOUSE 5). DE: OEFENSIN B. DE: CRYPTION 3 PRECURSOR, DEFENSN. HD. DFF7 MACME} ID: DEFC AFDAE E:NACACAMRAA DE: DEFENSIN C; INSECT EFENSN6 ARXEENSN-7 EFENSN DE (ANTIADRENOCORTICOTROPIN : DEF: OSE X: EFC ORNMO PEPTBE, (I) OE. CRYPTN-7 PRECRS OR DE: BEFENSIN. C. {FRAGMENT). DFFFNSIN I}): {}}EF3 RAT ED: DEFC RHOPR XERA NEP. PEPE }{: DEF7 RAB}: E: RENSEFENSES C. {RINP-3). RAF CORTICOSTAFEN DE: DEFENSN INSECT DEFENSINS) (R3), DEFENSIN
TO: DEF8 MoESF Ex: E AEA II): DEF4ANEDAC E: CRYP-8 PRECRSCR DE: EFENSN PRECRSR E ANBRCNS (ERAGMENI). DEFENSIN. (AAL3)EFED) (FRAGMEN3). ENSECT DEFENSIN.INSEC-EFENSEN EFENSIN LiKE II): DEF’9 MOCSE E. CRYPTEN-9 PRECURSOR ED: DEFD, ORN MO ID. Di F4 HR FMAN (FRAGMENT). OFFENSIN DE EFENSN DE. NEFRPHILEFENSIN 4 (HNP-4) (HP4). | D: DEFA. AEDAE. ED: DEFA ESCY DE: DEFENSNAPRECRSOR E: INSECEDEFENSN, EFA ESA (AABEF)(ARTHROPOD OEFENSIN). OE HAMSFRNEJBROPH O: OFFI A PFPTIDE 4 (HANP-4; NEUROPHIT ID: DEFA ANOCP DE. INSECT OFFENSN EFENSIN: OE: DEFENSIN A INSEC BEFENSEN. HD: DEFI APIME ID: DEF4 MOUSE DE: ROYALISIN, INSECT E: CRYPDN 4 PRECERSOR ID: DEFA MAMBR EFENSN. (FRAGMEN}}, OFFENSIN. DE: DEFENSEN A INSECT DEFENSN. II: DEFI BOMPA II: DEF4 RABIT E: ENSECEFENSN E: I}): DEFA MOSE (ANTIADRENOCORICOTROPIN DE TESTIS OEFENSINPRECURSOR II: DEFI DROME PEPTIDE III. (FRAGMENT). E: ENSECOEFENSN PREC:RSOR IE): DEF4 RAT E: ERA NERPEDE II: EDEFI PALPR (RTNP-4), RAF CORTECOSTATIN E: ENSECEFENSN. (R4). DEFENSIN II: DEFI PEELDU F: DEFS HE}MAN E: ENSEC ENSN. E: DEFENSNS PRECSRSR ID: DEFI PHOTE ID: DEFA REOPR DE:PORyCEN PRECRSOR IE): DEE'S MACME OE.RODNES DEFENSINSAAN (INSEC DEFENSENSA AN.E) B). BE: EACA CAMATA B (INSECTIOEFENSENS) EFENSIN. ANEFENSEN.S ID: DEFIPYRAP I: EEA AOPAI E: ENSEC DENSN IED: DEF5 MOUSE DE. NSEC DEFENSN, ISOFORMS DE: CRYPTEEN 5, DEFENSIN. BAND C. ID: DEFI TENMO E: ENECN PRECRSOR. IE: IEF5 RABET INSEC DEFENSN-LKE. DE: (MICROBICIAL PEPEEE NP II: DEFE, AELAE A}. DE: DEFENSNB: ENSEC {I}: })}EFN HELVI DEFENSN. DE: HELORICN, NSEC ID: DEF5 RAT DEFENSIN DE: IEFENSINS PRECURSOR RED : DEFE ANCP 5). DE: DEFENSIN BINSECT HD DEFN HUMAN OEFENSIN. Patent Application Publication Aug. 4, 2011 Sheet 15 of 28 US 2011/O1892.87 A1
FGURE 7H DE: DEF1 HEMAN, DEF2 HUMAN. E: EPERCN SERSR 3E: DEMASE IN E3 DEF3 HUMAN ENSEC DEFENEEEEEE {E): )MS4 PHY SA ID: DEFV MOISE ED: DISA PHYDI {}}E: DERMASEPTIN 4 (S. EV). DE: DEFENSIN-ERE PROEN 4C-5 EDE: ESA, CHAEN OF DESTINCTIN. PRECRSOR, HEERODMERC ANMICROBA IE): DMSS AGAAN PEPTOE BEEKMASEPEN AA-3-4 II: DEFW MOUSE PRECRSR E: CEFENSINKERENC E): i) ISB PHYi}} PRECSOR. DE: DESB, CHAIN 2 OF IISTINCTIN, }}}:{MSS FACIA EEROMERICANNICROBA E: ERMASEPTN P-3. IE): DEFX AAOUSE PEPE RECRSOR E: MOUSE DEFENSIN. CRYPEDEN RELAE PRON AC-4 II): DLP1, ORNAN ID: OMSS HYB. PRECRSOR (CRS4C). DE: DEFENSN-KEEPTDE DE: BERMASEPENB5. (DLP-1. IE): DEFY MOUSE iE: OMS5, PHYSA DE: CRYSTN-REATED 4C II): DLP2, ORN AN DE: DERMASEPTIN 5 DS V. PRECURSOR (CRS4C, DEFENSIN. E: DEFENSINIKE PEPTOE 2 (DLP-2). IE): DMS6 AGAAN IE). IEFZ MOUSE E: ERASEIN AA-3-6 DE CRYTON-REATEC ID: DLP3 ORNAN RECRS). PRECURSOR (CRSC). DEFENSIN. DE: BEFENSINKE PEPE 3 {{DI.F-3). IE): DMS6 PHYBE DEF ACA BE: DERAASEPINE DE: EFENSINE PRECORSOR HD. DMS PACE)A E: ERMASEPEN --5 IE): DMS7 PHYB: II); DEFAEE)AE PRECRSOR DE: DERMASEPTN BRG3 DE: DEFENSEN (FRAGMENT). PRECERSOR INSEC DEFENSIN Ei): i) MSt. PHYBE EDE: OERMASEPNB PRECURSOR B: OMSs PHYB} ID: DEF ANOCP DEPHYLOXN PRECRSOR: E: XEFENSINSAAN BNSEC E): DMS PHYSA DEASE INDERWE PEPE. EFENSN DE: )}. RMASE''EN (DSI) IE: OMYC DROME IE3: DEF CAVPO I}): DMS2 AGAAN DE BRBSOM RECRSOR DE: DEF1 CAVPO, DEF2 CAVPO E: ERMASEEN AA-2S E-RECHE FEFFIDE): INSECT (CORTICOSTATIC PEPETOE (F-CS) PRECRSOR. EEPE {CP-2}}. IE): DMS2 PACE)A is: BMYC DROII IE): DEF ERITE E: ERMASEPENP-2-2 E: EROSOMYCIN-LIKE PEPEEE E: NSEC EENSN PRECRSOR IE): DROS DROME IE: DEF GALME ID: DMS3. PHYBI DE: EROSOCIN PRECIRSOR: DE: ANNGA EPE D: ABENREG3N NSEC ENCEPP). GALLERIMYCIN, INSECT (DERMASEPSIN B2). DEFENSEN ?). IE): DTH HALAU ED: 3MS2 PHY SA 3E AN YNAREN ID: DEF LEQU EDE: DERMASEPE IN 2 (1}S II). iDE: SCORPION DEFENSIN (INSECT {E): EAFE EUCl3). iDEFENSIN-LIKE) HD. MS3 AGAAN 33E: BEAE-PAN ANEENGA E: ERMASEPNAA-3- PEPHE, PRECURSOR IE): A BOWN II: DEF ORYRH Ei). MSS PACA DE ENTERICBETA-DEFENSIN DE: DEFENSIN FRECRSOR PRECURSOR NSEE ENSN E): i) MS3. HYBE E: ERASEN B3 ID: EBCN BOMMO I}: BERB PHYBI DE EN39N NSEE BEENCE E: XERMAXN PREFRS) I}): DMS3 PHYSA EPE E: DERMASEFEN3 (DSEE). : ECA CA 13: Di'A PROBE IE): DMS4 AGA AN EC 3, HORSE MYEOD DE: DIPERICEN A INSECT E: DERASEEN AA-3-3 CATECBN 3 DEFENCE PEPE PRECRSOR E: ECAE2 EQUCA IE): DPD PHOTE !): DMS4. PACE)A DE: ECATH-2, HORSE MYELOL) DE: OPERCN PRECRSOR: E: ERMASEPN P-3-6 CAHECN 3 NSEC EFENCE EPE RECRSOR IE): ECAE3 EQUCA IE) IP DROME ID: MSA PHY BE Patent Application Publication Aug. 4, 2011 Sheet 16 of 28 US 2011/O1892.87 A1
FIGURE 7 DE: ECATH-3, HORSE MYELOI) CATHEICON 3 ID: ESC2B RANBE ED: HE SAISA ESCENIN-23. 3)E: HIS ONE H.E. (NB HE ID: ELAE HUMAN ACESSION NBERRVE DE: ANSI EGKOPRCEEINASE EE). FACE}) FACTR IS FOR SALMO GAIRDNERI) (SKALP). {DE: ACER, CSR CHELCERAEERENCE PROEN II); II2AX ONCSAY II: EMB CAVPO EE: HISTONE H2A (FRAGMENT). 3: ESINCP RAN.E. :FOR MYRG ASCEN PRECRES) E: FORAAECIN INSECE HD: EF2A BUFBG (MBP-1). ENCE PEPE OE: HISTONE H2A (CONTAINS: 8 FFORINS AND II) (FRAC MENT). ID: EMB2 CAVPO ID: FOR2 MYRGU DE: EOSINOFHEI GRANEMAOR DE: FORMAECJN 2, INSECT ED: H2A-HPHI BASPROEN 2 RECRSOR EFENEPEEE 3)E: HIS ONE H2A CONTAINS: (MBE-2. HIPPOSIN) (FRAGMENT). E); GAEL RANR; F: AFRN-. E}}: HALN HAS.A. E: CHANS OF HALOCEON ID: EMBP HUMAN HD; GAE2 RANR; 3. ASSAEE) 8) BASIC E: GAERN-2. HD. HDRN }{A}}AZ. PROTEEN). DE: ARDRORN }}); GAE3 RANRU IE). ENAP HORSE E: GABRN-3. ii). HEMI PYRAP SNAP-1 (ANTH3ACTERIAL EDE: EAEPERCN ENSEC PEPTIOE) GRANULN, ID: GAE4 RANRL DEFENCE PROTEIN NCEE E: EAEGKN-2. II: HEMO HYACE D: ENAP2 HORSE EE). (AES RANR; EE: HEMOLIN PRECURSOR (P4 : ANVCROBA, PEPE E AERN PROSEIN) (HEMOCYTE ENAP-2 (FRAGMENT. AGGREGATION HD; GAE6 RANR; INHIBITOR), INSECT DEFENCE E: ENK CWN OE GAERN-6. OEN SE: ENKEELY EN: DEREVIED FROME PRENKEPEAN. A. PRESS }}: GALN KASSE HD: HEMO MANSE {CONTAINS: DE ME}- IDE: GALENSEN PRECURSOR, FROG DE: HF MOI. IN PRECURSOR (P4 ENKEPIIALIN; LEU-ENKEPILALIN: ANACROBAL PEOE. PROTEIN) (IIEMOCYTE AGGREGAON ID: ENTA ENTDI ED: GAM ANOGA NHIBFOR), INSEC DE: NONPACENCORE. E MICINPETATIVE ANCROBALEEEE FRMING PEPEPRES) ENFECTION RESFONSIVE SHORE (APNP). DISPARPORE-A PEPFIDE PRECORSOR) : HEP HAN DE: ANTMICROBIAPEPSOE ID: ENT B ENT DI }}}: GKBE, GENBI HEPCIDIN PRECRSOR (LIVER BE PRE-FORMN KEN DE: GENKBILOBIN (GNL) EXPRESSE ANIMCE3A SOFORNB PRECERSO FRAGMENT). FEERIEDE. LEAF - BSPARPRE ID: GLOW HYACE ED: HEPC MORCH ID: ENTC ENTO E: GLOWERN. ENSEC DEFENCE EDE: HEPCON. EORE-FRANC PREN EE SFORMB PRECRSOR ID: HEPC MOUSE DSPARCRE-3. ED: GMAP43PRC CAVPO DE: OEPCON E: EAS MYE) E: ESA RANES ANIBACERAL PEPDE II); HEPCONCMY E: ESCENNA PRECURSOR: CAP RE: PUAWE HEPCON AECBN ANBAERA. EE ID: ESIB RANES (FRAGMENT. BE: ESCERN. EB PREFRSO. {{): HEPC PG ID: GMAP43 CAVPO OE, ANMICROBLAPEPOE 3D: ES] RANES E: GLINEAPG MYEO) HEC NEWER EXRESSED E: ESCENN ANTIBACERAL POLYPEP''.i).E. ( . KDA POLY FEFTIIDE CAF E IE): HEPC RAT iD: ES2A RANES DE: EPEOHEPCON. ESCENEN-2A {{). (NTY HiMAN DE: ACITVATION PROTEIN 519). II); IITIAP MYXGL IE: ES2B RANES E: HAGFISHINTESTNAL B. SCENN-B ANMCRO.B.A. P.E.S. BD: GOME ACAGO (HFEAP-E AND -2). CRANLATE ID: ES2 RANLU OE GMESNAREEROPD EEERSE PEPE B: SCNN. OEFENSEEIDE (CAIHEE ICED-IN). Patent Application Publication Aug. 4, 2011 Sheet 17 of 28 US 2011/O1892.87 A1
FIGURE 7J (FALL39, L.37. IE): HFIAP3 MYXGil IE): NDPRCBOVEN HCAPE8}(CATHEELICIN). E: HAGFSH NESNA ENOOCON ANTIMICRCBEAL PEPEIDE (HFIAP PRECURSOR(CATHEICIDIN) D: 3 HMAN 3. CRANIAEEEEFENSE PEFIDE DE: HCAP-8, FALL-39, L-37. (CAEHELICEDEN) ID: IND BOVIN DE: NOOLECON BD: LLP. HIV ID: HIS1. HUMAN DE: LLP (LENTIVIRUSIYEEC DE: HSEAN PRECFRSOR ID: ESC OPCMA PEP), (HESTIDENE-RCH PROTEIN 1) DE: ESC CYOOXC SCORPION (POSI-PB PROTEIN) (PPB). PEPE 3); E.I.P EIAv DE: ...I.P ENTIVIRUS YTIC II: HIS MACFA {{): AP RANJA PEPTIDE) E: HSAN . BEAPONCN {{D: LLP SV IE: HIS3 } EUMAN {E}: JAP2 RAN3A BE: }LP (LENTIVIRUS YTIC E: SAN3 P&ECRSR BE:APONCEN-2. FEEIDE) (HISITEDENE-RECH PROTEIN3 (PB) (BASIC HISTOINE RECH PROTEIN) IE): KABI. O. DAF (CONIAENS. HIS. AiNS - () 12), XE: K.A.A.A. B. is): EXI YCCA E. CXN s: HSS HR MAN : AP BOWN DE: HISTATIN 5 (HISTLINE-RICH DE: NGA ANTICROBA BD: LEX2 YCCA PROTEINS5) (FB) (BASIC PEPEEE PRECURSORBOVINE DE EYCOBX 2. HISTIDINE-RICEE PROTEIN3. BEA-EFENSN. I}): LUML. L. MRF ID; HLT HUMAN }}): LBP H MAN DE: EMBRECEN WRM B. ACOFERRN FRAGMEN POPYSACCHRE EFENSE PEPE HL AND 2 BNN, PROEN PRERSOR, {{D: LYS EISFO HC2. His O: B.B RAB, DE: FETION, I.YSENIN-RELATEI) E: IOLORICIN 2 PRECURSOR: E: PPCSACCARDE PROTEIN (HEMOLYSEN). INSEC EFENCE PEPEDE; BNNGPROEN PRECRSOR COLEOPERC (LBP). IE): MACE) LETGE BE: MACFLATEN ... AMPHEBAN {E}: }_{EB BOVIN DEFENCE PEPEEDE IE): HO! 3. HOLDE 33: ACERRCN 3 FA F: HO}(RCIN 3 PRECIRSOR, FRAGMENTS }}). MAC 2 LTGE, INSECT DEFENCE, PERE, (LACTOFERRIN/LACTOTRANSFER DE: MACAN 2, AMPHBAN CCEERCN RIN FRAGMENTS). SEFENCE FEPE ID: HPA RANES ID: LCFH HUMAN }}): MAC2: LITGE E: EMLYBEC PRENA DEAOFERRON DE: AACUATN 2 AMEBAN FRAGMENT). (LACOFERRIN FRAGMENT). DEFENCE PEPE li): HP39 RANES ii): CRPPRIMA {}): MAC3. IFGE OF HEMOYTEC PROTEIN B9 OS CORCOSTATN-REATED DE: MACULATIN 3. AMPHIBIAN {FRAGMENT). PROTEIN LCRP. EFENCE PEPTOE IE): LEAP2 PIG {D: MAGE XENLA ID; HRCYC HUMAN BE: NAGANN E: RECYCLIN IE): } EB1, BOMMO {}}E: EEOCEN i 2 & 3CRSs)R. EE): MAG2 XEN). A IO: HYP ARAEY NSEC ANTMCREBAL PRCEN BE: MAS ANN DE: HYPOGEN: PLANT ANFNGAPPOYPEPE E: EB3 BOMMO byAGA XENA (FRAGMENT) IDE. LEBOCN 3; INSEC DEFENCE DE MAGAINNS PRECRSOR. EEE ID: HYA AEIME BD: MAP34 CAPH DE: HYMENGPAECN INSEC ID: LEB4 BOMMO DE: GAOAT MAP34-A (MAF34-B); DEFENCE EPIDE DEEB)CN 4 PRECFRSO FERCM (AELEN. GENE ENSEC ANTIMICROBAE ii): IAAT MAIZE PREN 3): MAST POIJA ... APHA-AMYASERYPSN DE: POSTES MAS (PARAN. INHIBITOR PLANT ANTIFUNGA : LEV. XENLA PROTEIN). O3: PROLEWISOE PRECURSOR II); MAST VESBA E: EASOFARAN ID: BAMP IMPBA {{): LLS7 PRC HUMAN E: ARTMCRBA PEPRES E. 9 KOPOYSACCARDE IE): MAST VESCR PREC, FRS(R: BAMP-EBAMP4, NBNS PEN PRECRSOR BE: MASPARAN . PAN ENE PES Patent Application Publication Aug. 4, 2011 Sheet 18 of 28 US 2011/O1892.87 A1
FGURE 7K IE): NCC, RANNI ID: MAS, VESLE {{}: Mk1 PAEPR XENRCN EASOPARAN DE: MiEA NIROWN is INSECT DEFENCE REPEDE ID:NGC2 RANNE : MAS WSM3 E: NORCN DE: MASYOPARAN M (MASE CELL Dw&A. PAPER DEGRAN FLATING EEEEEE). ALNKOWNEE-A ENSEC ID: NKK PIG EFENCE EPE E: NK-YSN ID: MAST VESOR DE: MASIOFARAN HISTAMENE II: MK2B PALPR IE): NKK2. PEG RELEASING PERFIDF I) (HR-9). E. v.FTALNIKOWN 1-3, NSEC: E: NX-YSN 2. OEFENCE PERTOE ID: MASE WESXA ID:NKL EQUCA F. MASPARAN X. T}; MK3 PAEPR ANWR3A EENK DE: MEANIROWN INSEC YSN. ID: MAX! BOMMA DEFENCE FEPOE E. MAXIMN FRECURSCR :NK, PC; CONTAINS: MAXIMIN... ID: MRCN BOMMO DE: NK-LYSIN FRECEIRSOR{NKL EORNS AN 2 (FRAGMENT. PORE-FORMING II: MAX2 BOMMA PRECEIRSOR (INSECT DEFENCE EEE DE: MAXIMN 2 PRECURSOR PEPTIDE}(NA&EE CONFLICE) CONTAINS: MAXIMIN-2. ID: NPA NAEFO R. RATA DE:NAEGERAPORE A PRE ID: MAX3 BOMMA DE: FRGMETE-REAED FDRING EPE DEMAXN 3 PRECISO PEPTEDE (MRP) {CONTAINS: MAXIMIN-3. ID: NPB NAEFO II); MSGP MOSE OENAER.ERAPOREBPORE ID: MAX4 BOMMA DE: MEYEIOOSECONOARY FORMING REPEEDE. E: AXMN 4 RECRSR GRANULE PROTEIN (MRNA CONTAINS: MAXIMIN-4: (CLONE B6. IE): OELPA EOBAC E: OSMEON-LKEPROEN ID: MAX5 BOMMA IBD. MK DROME PRECURSOR PATHOGENESIS DE: MAXIMNS PRECURSOR OE.; SAETCHNIKOWN RECORSOR: RELATED PROTEIN PR-51D). CONTAINS: MAXIMIN-5. NSC FENCE EP ID: ONCIII (NCMY ID: MAX6 BOMMA ID: MYCAMYTGA E: ESCBNEE6-KE PROEN DE: AXEMN-6. DE: MYFICIN A PREC:RSOR, FRAGMENT). MLSCEFENSEEE ID: MAX7 BOMMA ID: ONCE ONCMY DE: MAXIM.N.T. DE: III. ID: MYCB MYTGA II: MAX8 BOMMA IDE: MYTICINB PRECURSOR, IE): OFoE OXYK E MAXIMEN-8. MSC). ENE EXE DE: OXYOPININ } {OXKIE). ID: MBP MAIZE IB); MYMY AAYTEE) B: ANMRAEE IDE: MYTIMYCIN (FRAGMENT). I}): OP2 OXYK MBP-PLANTIEFENCE PEPTIEDE DE: OXYFININ 22A, 2B, 2C& 23). IO. MYTA MYTED ID: MCDP APEME E: MY IN A Mil. USC I}): OPO OPECA OEMAS CELDEGRANULANG EFENCE PEPEDE DE; OPSECPORN & PEPTIDE (MCD) (PEPTEDE 40). B: MYB MYE) IE): P15A R ABET II. MCDP MECPE IDEMYFILINE, MUELCSC E: 5 KEXPROEN ARECRS OR E: WAS CEERANTANG DEFENCE EEE (F5A). PEPEDE (MCD). IBD, MYEB MYEGA ID: P58 RA81; ID: MEL APIME OE: MNB: y ESC E: S KO PROENB PRECRSCR DE MEN AC) DEFENCE PEPE (P15B). FRERSCR ID: MYFC MYFGA ID: PAFP PHYAM ID MEL2 APIME E: MY IN C. M. I. FSC DE: A.N.I.F.E.N.GAL PROEN E OEMIEN MINOR EFENCE PEPEE PRECURSOR (PAFP-S, AFPS-}. PLAN DEFENSE PETE II: ME) APIC BYO MYSA E: EIN. DEMYTILIN; M:ISC ID: PALC RANPA DEFENCE EEE E: PASTRN. C. NSN II): MAPEF, REASNCS PEPE). DE: METIN. ID: MYEG MYTGA OEMYTLENG: MLSC IE): PAN ANIM. ID: MIS MESAN DEFENCE PEPTOE DE: ANONIN i ; SORPRION DEMSGRN WENOAA ANMICROBA PEPTOE Patent Application Publication Aug. 4, 2011 Sheet 19 of 28 US 2011/O1892.87 A1
FGURE 7. ED: PDEFI, VICFA II. PAN2 PANIM II: PEREF BRANA EF: AFAN PANT OFFENSIN EPANONN : SCORPRON E: CYS-CHANREPUNGA WENON ANMCRBIAL PERTIE, PROTEIN (AFP) P:AN ID: PEF VIGRA DEFENSIN (FRAGMENT). IDE: PLANT DEFENSN (PUTARIVE) ID: PAR PARAS DEPARASIN (HESTONE 2A E: PEDEF1 BRARA DERIVE DEEENSE PEPTIDE). E: CYS-RCE ANEENGA : PEF WEA PROSEIN (AFP) 8:AN } DE: AMA-PERBEENN ID PARDX PARMA EFENSIN FRAGMEN). (THG-PLAN DEFENSIN. XE: PARAX. E. PEDEF CACA ID: PEEF2A S.INAL ID: PARDX PARPV E: FIAN EFENSNS OE: CYSRC ANTNGA XE: PAREXAXNSP-P-2 AN is P3 (Pt:IAWE). PROTEIN 2 (AFP2A) (M2A)PAN: DEFENSIN BPO PARSC : PEF CAPAN E: PARA3PRN: BMKBP. DE: EFENSIN - PRERSDR ID: PEF2B.SINAL E. CS-RCHANFNGA IE); CG PACGO i. PDEF CE: E PROTEIN 2B (APP2B (M2B) PAN: E: NERCN DE. C.SS.RICE ANEFENAL EFENSN. PEOPEIN (CT-AMP). EEANT IE): PC(2 PACGO OENSN }I): 'BEF2 ARATH E: CNEN E: AE ANEENGA i): PDEF) DAH&E PROTEIN (CYS EINE-RICEE IE). PC(3 PACGC) DE (YSRCEANRNA ANEEFUNGAL PROEEN), FLANT E: ONERCN (3. PROTEIN (BM-AMP), P.AN} DEFENSN DEFENSIN II). PDEF2 BEVU CG4 ACGO y:PDEF HESA OEANFUNGA ROTEN AX2. E: ONERCNG. E: CYS-RCHANTEUNGA PROTEIN (HS-AFP, PIANT ID. POEF2 BRANA li); PCG5 PACGO EPNSN E: CYS-RCHANFNGA DEPONERCN G5 PROTEEN 2 (AFP2), PLANT 3: IEEE ORSSA EDEFENSIN FRAG&ENT). ID: PCG6 PACCO DE: PLANI DEFENSEN (PUTATIVE) XE: PNERCNG6 ED PEF2 BRARA {}: PEDEF 'EHY E: YSRICANNA I}). PCG7 PACGO E: FORADEFENSN-IKE ROEN 2 (AFP2) PIANT XE: PCNERCN G. PROE PRECRSCR IDEFENSIN FRAcav ENT). (PHDF)(PLANT EFENSIN) IE PCL: PACGC) ID: PEEF2 CAPAN DE: PONERCN. B. PDEF PECGI. DE: DEFENSN-2 RERSDR DE: PLANT DEFENSIN (PUTATIVE IE): PCWPACGO BE: PONERCN W. ID: PDEF2 PETHY iD. PEDEF PISSA E: FLORAL DEFENSN-LIRE IE): PCW3 PACGO OE ESEASERESSEANCE PROTEIN 2 PRECURSOR (PEED2. E: ONERCNW3. ESPONSEROEN39 ERSOEAN BEENSN }I): {{}}EF2 {{SSA IE). PCW4 PACGo E CYS-ANFNCSA E: ENERN W3. PEF RASA PROTEN (n 1230), P AN: DEFENSN RE: (SRKEANRNCA II: PCWS PACGo PROTEIN E (RS-AFP1), PIANT II): PDEF2 RAPSA E: EPONERCNWS. DEFENSN OECYS-RICANNGA PROTEIN (RS-AFP2), FLANT ID: PCW6 PACGO H): PDEF SNA, OFENSIN E: CNERCNW6. E: CYS-RICHANTEFUNGA PROTEIN (AFPi)i&ii) PLANT HD. PDEF2. SORBI ID: PDEF1. AESHI EFENSN E. CS-RCHANFENGA E. YS-RCHANTFNGAL PROTEIN (SI-ALFA-2), PLANT PROTEIN E (AH-AMP, PIANT D: PEDFF SORRE FENSN EESN E: SEAL PROEN ENEBOR E ENSEAPAAAYASES
ID: PDEF1 ARATE (ST ALPHA-}}. D: PDE2 SPOEL XEAN A PEEEDEPOFE E: YS-RE ANFRENA PAN DEEENCE PEPE T. POEF SPIO), FROTEIN 2 SO-2) EEAN E: YSRCHANNGA DEFENSN IE: PLEF). BEFVU PROEETN 2 (SO-3) PLANT DE: ANTNGA. PRCENAX DEFENSNFRAGMENT ID: PDEF2 VICFA PAN XEFENSN i)}: ABA''IN-2 (PLAN: DEFENSIN) Patent Application Publication Aug. 4, 2011 Sheet 20 of 28 US 2011/O1892.87 A1
FGURE 7M E: YS-RECAN FNA II): PDEF2 WEiEAT FROTEIN (GAMMA1-EB, PLAN : it): PFPA ENTH DE: Gamma-2 purchionin (THG-2), EENSN : AACRE OR. RANTOEFENSIN. FORMENG PEPE SFORMA ID: PDEF MAIZE ID: PIEF3 ARATH DE: GAMMA-ZEATHEONIN 1 THG) B: FEB EN THT DE: GAAMA-HONIN HONOLOG PANDEFENSIN. DE: AMBEBAPORE POR AT2G0210) PRECORSOR (THG). RVNS P. SRM PEANT DEFENSIN (PLFATIVE) II): PDEF MEDSA : ANNG > {{D: PFP ENIHI II): PDEF3 BRARA PRECURSOR (AFI). PANT E: AREBAORE ORE DE: CYS-RCHANIFNGA EFENSN, FORMENG PEPOESORMC, PROTEIN3 (AFP3) PANT DEFENSIN (PUATIVE). D: FFR PEG ID: FOEF NICAL XE: DESECF DEFENSN DE: EROFHENINS PRECORSOR (EF ID: PDEF3 PESSA PRECURSOR NAD). (CLONEC 12). Pi-2)(CLONE DE. (ANTIFUNGAL PROTEIN PSD). C6}{CATHELICIDIN). II): PDEF NICFA It): PDEF3 SORBI E: AyA-CNN {{D: PFPENFH: E: PLANEEFENSIN PRECURSOR (THC- PELANT E: PERMING PEPE DEENSN, PRECURSOR (CEH-APP). y: PE3 SO DECSRHATFNGA ID: PDEF NICTA BD: FF PIG PROFEIN 2 (SO-D3) PLANE DE: CYS-RE AN ENGA i)E: 'ROFHENIN (PF-1, -2).(CLONE EDEFENSIN, FRAGMENT PROTEIN (PST), PLANT OEFENSIN C6AN C2). II: PDEF4 ARATH D: PEF EN {{); P: PIG DE: OAMMA-IONNOMOLOG E: CYS-RECANFiNiCAL OE: PROTEGRIN (PG-1). AT2G02)2O PRECURSORCEHC-2); PROFEEN (''}}. PLANE DEFENS EN PANT DEFENSIN IED: PG2 PIG ID: PEF SOLIU BE: FROFEGRIN 2 (PG-2). ID: PTEF4 FISSA E: CYS-RANTENGA DE: EFENSE-RELAED PEPIDE 2 PROTEIN (P322), PLANT DEFENSIN (DEFENSEN2) (ANTEFUNGA : G3 PG PROTEIN PSID2), It): PDEF SOYBN DE: PROTEGRIN3 (PG-3). DE: PROTEINASE INHIBITOR P322). It): PDEF4 SPOi. PAN DEFENSN {{): P(4 PIG DE: CYS-RICHANIFNCA EE PROFEGRIN APG-4). PROTEIN 2 (SO-DS, PLANT DEFENSIN, FRAGMENT by CSS ID: FBEF ERIAE DE: PROTEGRINS (PG-5), DE: CYS-RCHANIENGA PROTEIN (GAMMA -P), PLANT {{): PGK XENLA II: PDEF5 SPOil EENSIN E: EWDE RECERS E: CYS-RCHANIFNA FRAGMENE (ANTEMCROE3AL PROTEIN 2 (SO-D6) PLANT ID: PDEF VIGUN FEERIDE PGK). iDEFENSIN, FRAGMENT BE: CYS.R.CH ANFNGA PROTEIN (PSASO), PLANT }}). FGFA XENLA ID: FIDEFG SEO, DEFENSN DE: ANACRBA PEPE E. CS-ANNSA PA PROTEIN 2 (SO-D7) PLANE ID: PBEF, WASJA }}EFENSIN, FRAGMENT (AyMA-CNN ): PRC PG PRECRSOR. OE: PROTEGRINS PRECURSOR (PC lf): PDEE7 SPIO}. - PG-5)(CAE HELICIDENS). DE: GAMMA-E HONIN 5 (DEFENSIN ID: PEPARANCA. D5) (FRAGMENT). DE: PEPSINOGENA DERWEED BD: PGQ XENLA PEP II)}, (BFAAP) E ANTMCRBA PEPE ID: PIEFA EIELAN E LOWER-SPECC KAMA-. ID: PEPCRANCA THIONIN PRECURSOR GDEFENSIN DE: EPSINOGEN-CDERVE II); PIP RANP SD2). PEPFIDE (b?cAF) E: PPNN, BREWINN-FA ID: PDEF8 HELAN IE): PERE MOUSE II); PSE MORSA DE: DEFENSIN (FRAGMENT). DE: PERFOREN 1 PRECURSOR (Pi) DE: PISCON MORONECEN (LYMPHOCYTE PORE FORMING P EAN PROEN). is S3 MRSA DE: BE 3NSIN CLA (FRAGMEN}). DE SEN 3 ID: PF4PF HOMAN II): PDFF HORVF DE: PATE.E. PACE OR 4 (PF-4 ID: PLEO. A PLEAM. PEIDE FRAGMENT, Patent Application Publication Aug. 4, 2011 Sheet 21 of 28 US 2011/O1892.87 A1
FIGURE 7N E: ERONECTES AMERICANS DEPG MYEO) DE PMONARY-Si3RACANS PLEUROCON-LIKE ANTIESACE ERAL PEPEIDE (EMAP PROTEIN). PREPROPOLYPEPTIDE(PI E1A) 37). H. P. RF PEG IO: PLEOf HIPPL ID: PN ENVA E: FRV CNE 6C E EROCON-KEEEEE DE: PENAEIDIN-E (Pi), (CATHELICBIN). A. CROSACEAN DEFENCE EPE PA. WHEAT II); Pi Foi PL FAM ID: PN2 PENVA DE: PRINOOLNE-A XE: PERONECESAMERICANS iDE: PENAEE)IN-2 PRECURSOR (P2), PRECURSORFIAN EFENCE ERON-E CRESTAEAN EFENCEEE RROEN PREPROPOLYPEPIDE(PLE1) {}: PN3A PENVA H. PUIB WIHEAT II); PLEO2. HIPP) E: PENAENE-3APRERSR DE PERNONE E: EROCON-LIKEPEEDE (P3-A, CRESTACEAN DEFENCE PRECBRSOR, AF2, FEPOE ED: PYLA XENTA ID: PLEO2 PLEAM ID: PN3B PENVA DE: PYAGLA PRECRSOR, E: PLORONECES AMERICANS E: PENAEIN-3B PRECERSOR PEROCON-KE (P3-B, CRYSTACEAN DEFENCE D: PYRR PYRAP PREPROFOLYPEFIDE(PLE2) EPIDE EDE: PYRKOCORIEN INSECT DEEENCIEPEPTOE ID: PLEO3 HIPP). ID: PN3C PENVA DE: PLEEROCON-IKE PEPEEDE E: PENAERON-3C PRECRSOR II); RGSNA RANR; A3. (P3-C). CRUSEACEAN DEFENCE DERGSINA PEPE It): PLE03 PLEAM II. RGSNB RANRI BE: PLERNECES ARERICANS ID: PNAME HANA E. RiiOSNB. PLEROCON-KE EANTCROBAL PEPTEPN PREPROFOLYPEPIDE(PLE3) AMF (PN-AME FN-AMP2), PLANE ED: RGSNC RANRU DEFENCE PEPEEE DERGCSN C. II); PEE04 PLEAM E PLERONECES AMERICANS IO: PP PHES II: RHIC ORYRH PLEROCONKE DE: ANEMCROBAL-KE DE RHINCCEROSIN FREC RSOR: PREPROPOLYPEPTIDEOPLE4) PEPTOEPP INSECT DEFENCE PEPTIOE, CEOPTERCNE ID: P E PEAM ID: PPME LIMPO BE: PLEOROCON OE: POLYHEMESAN I. EL): RK RABI: CHELICERATE ANMICROBA ERA KNEY PEPTOE CRECOSAFEFFNSN-KF l); PMAP23PRC PEG EPBE RK EPGAYEC ANBACERAL PEPE II: RL37 MACMU P:RECURSOR (PMAP-23, MP23) DEK-37. (CATHELICIDN). ID: PPM2 LIMPO OE: POLYPESN II: RLXN RANCA II). PMAP23 FEG CELERAE ANCRCBA EE: RANALEXN PRECURSOR E: PG MYEO PEPE ANTIBACTERIAL PEPTIDE (PMAP ID: RNXEC, RANGR 23). IBD: PR39PRC PEG OE, RANALEXN DE: ANTBACTERA, PEEPR II); PMAP36PRC FEG 39 PREC FRSOR (CATHELICIOIN) II: REGA RANGR E: PVYEOD DE: RANAUERINGA ANTBACERA, PEPTE ID: FR39 PEG PREC FRSOR (PMAP-36, DE: ANTBACTERAL PEPSBE PR ED; RTGB RANGR MF36(CATHELICIEIN) 39. E: RANARN-GE ID: PMAP36 PEG l}); PSE PSEPA ED: R2B RANBE BE: GED ESEBN EDE: RANATERN-23. ANSIBAC; ERAL PEPTEDE (PMAP 36), II: PSE2 PSEPA II: Ri2GB RANGR E: PSEN-2 E: RANAERN-2B ID: PMAP37PRC PIG DE; PGAYEO) ID: PSE3, ESEPA ED: RE2: A RANAC ANIBACERA, PEPE DE: PSEN-3 OE: RANATERN-2A PRECURSOR (PMAP-37. MP37) (CAE HELICEDEN) ID: PSE-3 PSEPA Hy: Ri2}B RANLY E: FSEEN-4, E. RANAERN-23. ID: PMAF37 PG ID: PSPB IIUMAN II: RT2FRA RANTA E: RANAFRN-ERA. Patent Application Publication Aug. 4, 2011 Sheet 22 of 28 US 2011/O1892.87 A1
FIGURE 7O BESTYELIN A (FRAGMENT), {D. RE4 RANCA ID: SNA2 SOLU NCATE, DEFENCE, PEPTOE E: RANAER. 4. DE: SNAKIN2 PRECURSOR STSN2. (ED: STYB SEYC. O: SAC CNCMY ): S.P.A.M. PNSY EE: SYEEN B (FRAGMEN3. DE: SALMOC{{}}N (FRAGMEN'E). OE ANACROE3AEFEEE NCAEEEFENCE EEE PRECURSORs {S}P-AMP to SE }}); SABRK SAR1E AMP3: PAN'{}}EFENCH, PH.P. DEs IE): SEYCSTYCL E: SAPECNB 7R-7KFRAGMENT SYNC: NCA, SEC EEENEPEEE. ID SPD: A HYACE EENCE PEPE DE: SPOSA. INSEC {E}: SAPB SARFE DEFENCE PERIE ID: STYD STYCL E. SAPECIN B, INSEC EFENSTN. CE: STYELND UNICATE ID: SPD: B. HYACE BEFENCE PEPE ID; SAPCSARPE DE: SPODPSN EB, INSECT DE: SAPECINC, NSEC DEFENSIN. EEENE PEPE 3: AC EAC3 3E: ACEHYLESEN: H: SAFE SARPE. HD. SF BOVIN CHECERAE DEFENCE FEDE DE: SAPECIN PRECURSOR, INSECF DESEMNA. PASMN FRAGMENT(SPF), (CAITRTN BD: TAC TACIR FRAGMENT) DE: ACHPLESN REC:RSOR {{). SCAR (RHYR ECERAE ANMC8OBEA E:SCARABAECNNSEC ID: SPF2 BOVIN PEPOE EFENCE PEPTOE ESENAPLASMN FRAGMENT 2(SPF), (CALTRIN IE): TAC2 TACTR D: SCRP PANM FRAGMENT). BE: ACEYFILESEN PRECRSOR E. SPNE RECRSOR, CECERAE BEFENCEEEEEO II): SIG SEE:S }}): S}:CP APIME ESPNSNRN. it: AC3 ACG E: SECAPIN, INSEC VENOM E. ACHYPESN: NPEN ID SPYY PHYR EERAE ESENCEPEPE DE: SKNOYEDEYS {{). SHEP CAPBO (NEUROPEPTEDEPANCREATIC BD: TACA1 EACTR E: ANCROBA FEDE POLYPEPTHEYY ANALOGUE) EACHYSATNA SHEP-GRP. SHEPHERN (CHELICERATE ANTIMICROBIAL ID: SRIA SARPE PEP I HDE id: SEL: RH EP BE SARCOXINA RECRSR DE SELE:CN in GCS ANMCROBA PEEE {{): TACB 3 ACTR (). SRB SARPE, E. ACTSTATN D ARCCXNB PRECERSR (CHE CERAFE ANSIMICROBEAL INSEC EFENCEEEEEE PEPTDE) II. SIN BOVEN ID: SREC SARPE BD: TACC EACER DE SECRETOLYTN. DE: SARCOTOXINIC, INSECT DEACHYSEAN C SECRETOGRANN EFENCE PEPETE (CHELICERATE ANTIMICROBIAL (CHROMOGRANIN PEPTOE BANILE ACTERIAL FRAGMENT SR SARPE. DE: SARCOOXINE, NSEC {{): TACN TACR ONCEPTE {}}E TACITY CITIN: Ci}}...ICERATE 1): SMAP29PRCSIEEP DNC, PEPE E: SEEP WYEC ID: SR2 SARPE ANEACERAEPE OE SARCOEOXIN - SECRESOR Ba: TAP EOVIN PRECURSOR (SMAP-29, NSECREFENCEEPDE E: ERACHEANTICROBA MP29(CABHELICIDIN). CATHELIN PEPTIDE PRECURSORTAP), RELATED PEPIDESC5 ID: SR22 SARPE BOWNE BEA-EFENSN E: SARCOXIN-2 PECRSOR. D:SMA-29 SEHEEP NSECE DEFENCE. PEPE {{}: TCBE TRELO DE SEEME)) BE: RCCNCN BANE3). AN BAC; HRA, JPE}}}}}}, SC5, II). SR23 SARPF SMAP-29 D}: SARCO (XN I-3 REC, FRSOR, II: TDEF IAMACM: INSECEFENCEROEN DE: HEADEFENSIN-1, SFRENIT {{): SMAP34 SHEEE A PRECCRSCR (RTE)-. E SEEP YEC ID: SR2 SARPE ANTMICROBAEPE DE: SARCOTOXIN IAPRECRSOR, BD: TOEF).B MACMU PRECURSOR (MAP34}{CAIHELICID NSECTDEFENCE PEPDE DE: THETA DEFENSIN-1, SUBUNIT IN) B PRECURSOR (RTD-1). II); SOX SEOMA D: SNAE SOLIt BOE SCW(XN. IE): DEFE MACME} DE: SNAKIN-E (FRAGMEN), OE: RHESE IS THETA DEFENSIN PANT DEENC, PPF II); SIYASYCL RT. CYCC DEFENSE PEPTER Patent Application Publication Aug. 4, 2011 Sheet 23 of 28 US 2011/O1892.87 A1
FIGURE 7P DE: PANCREASTATIN: WE-4: li): E.A.RAN3A ED. PM. YCES CA: ESEAETN: E: EEMEPORN-AFROG DE: OSMOTIN-LRE PROTEN P'y. ANIMICROBALETE, | PREC FRSOR (FRAGMENT). I}}: XENOXENLA PANEFENCE. PEPRDE E: XENOPSINEXPRECURSOR AMBANEFENEEPE II: TEN3 TENMO ED: RFE CHICK E: ENECN 3 PRECURSOR, DE: OWGRANSFERRN ID; XT XENTR NSECT DEFENCE PETE PRECURSOR (CONAi ROMEIN) E: ANARE3A, PEPEDE {ALLERGEN GAL D 3) (GAL DIII). (XF-1). ID: 3 ERN PSEUS E: ERMICN FD. P2 PEN Ji. XF2 XEN:R DE: FEERIN 2. DE: ANTIMICROBAL PEPTDE 2 II); E LAN POIDMA (XE-2). IDE: HANATIN ENSECT BEFENCE | D: P22 PEN PEPEE. OE, PERN 2.2. ID. XT3 XENTR DE: ANTMICROBAL PEPE3 TF): "HHRHOR vi II: F23 PEN (XE-3) ( EVETDF-FIRE PEPE1 3F). BEANFNGA PROTENR E: FSPERN 23. (FRAGMENT), PLANE DEFENCE ID XT4 XENTR EP) ID: CP24 OPEN E. ANMICROBA, PEPIE 4. E: ERN2. XE-4) IE): EHESHORVU DE: ANFNGAL PROTENS ID: P25 FPEN D: XTS XENTR (FRAGMENT), PLAN DEFENCE DE PERN 25. E ANCRCBEAEPE is PEPE (X3-5 (PGLA-LIKE PEPIEDE). II. P26 JPEM. TE: EIN HOPEI DE: FERN26. II: X & XENER BE: GERINN DE: ANIMICROBAEPE is ID, UP27 UPEMJ (XT-6). ID: IN2 HQPTI B.E. RIPERN 2.7, E: GERNIN IL): F28 UPEM. B: Xii XENTR IE): 3IN3 IOFII DE: EPERN 28. O ANIMICROBAEPE 7 E: ERNN-3 (XI-7.
| D: GP36 UPEXi. ED: {TN4 HOPT DE, FERN 36. 3AM MAIZE BEEGERINN-4, : AEAMAN PRECRSOR: ED: UPS PEN LANTEFENCE ROTEIN ID: MPOB RANOR IDE: EPERINS 3, 3.2 & 3.3 E: BREWINN-OB. II. F3 UPEM. : MOC RANCR RF: FERINS 34, 3.5. AND 3.7 DE EMPORN-OC. ED: CP4 OPEN D: MOB RANCR DE: ERN 4. E: EMPORN-OD, ID: {P6 (PEIN ID: 3 MPGA RANGR DE PERN 8...AN 82 BE: EMPORN-1GA II: E71. I} Ew TD; $ MP A RAN DE: FEERIN 7, CONTAINS: E: EPCRN-A. UPERN 7.l. i. O: TMPLB RANi U ED: WIC 2 MACEN E: EADRN-B DE: WINSERSOR (MEASAF2), PLANT ID: MPLC RANLU ANMICROBAL PROTEN E: EMPORN-LC, E. WCS MACN IE); EMF, RATAG DE: WICILIN PRECURSOR MIAMP2) BE: EMPORN PANT ANIMICROBA PROEN II: RMPA RANTE ID, VIP PIG E: EvoN A. DE: (VIP) (FRAGMENT). IE): 3 MPB RANTE ED: VERE HELV E: EMPORNE OE: WRESEN II); MPL RAN''E I). VS : N BOVEN E: EARNL Patent Application Publication Aug. 4, 2011 Sheet 24 of 28 US 2011/O1892.87 A1
FIGURE 8
3.333 3.3: 3:38 83.88:8
&: $888: 8 fi:38 ::g: Patent Application Publication Aug. 4, 2011 Sheet 25 of 28 US 2011/01892.87 A1
FGURE 9
3. 8. & 88. 8::8.g
8.83 88
& 388 88.388 88.3 :8888.3 :::::: 8:8 &8:38.8 888 38883: 3:38. 8:8; 88 8888; 8: 8888; 8: 8888; &g 8888; 8: 888: 88 8 888 Patent Application Publication Aug. 4, 2011 Sheet 26 of 28 US 2011/O1892.87 A1
FIGURE 10
Patent Application Publication Aug. 4, 2011 Sheet 27 of 28 US 2011/O1892.87 A1
FGURE
PDMS stamp
Flexible secondary substrate
Patent Application Publication Aug. 4, 2011 Sheet 28 of 28 US 2011/O1892.87 A1
FIGURE 2
Six: {:
&
PE is S. Cry E.
Hair y - Epidermis Dernis US 2011/O 1892.87 A1 Aug. 4, 2011
METHODS AND COMPOSITIONS FOR ulcers, 0.5% from pressure ulcers, and 5% to 10% of people WOUND HEALING with diabetes experience neuropathic ulcers. The economic impact of chronic wounds for these conditions alone in the United States has been estimated to be well over S15 billion, 0001. This application claims the benefit of U.S. Prov. annually. With the population growing older, cases of diabe Appl. 61/303,414, filed Feb. 10, 2010, and is a continuation tes mellitus will increase as will the magnitude of the problem in-part of U.S. patent application Ser. No. 12/363,044, filed associated with chronic wounds in these patients. Jan. 30, 2009, which claims the benefit of U.S. Prov. Appl. 0005 Normal wound healing is an enormously complex 61/024,725, filed Jan. 30, 2008, the entire contents of which process involving the coordinated interplay between fibro are incorporated herein by reference. blasts, vascular cells, extracellular matrix and epithelial cells to result in a seamless progression through an inflammatory FIELD OF THE INVENTION reaction, wound repair, contracture and coverage by an epi thelial barrier. However, in many patients, due to either the 0002 The present invention relates to methods and com local wound environment or systemic disease or other factors, positions for the modulation of wound healing. In particular, the wound healing processes can become asynchronous (i.e., the present invention relates to promoting and enhancing loss of connectivity with triggering mechanisms associated wound healing by changing the intrinsic chemical composi with prior cellular events) and are unable to progress to clo tion and/or physical features of the wound bed. Sure, resulting in a chronic ulcer. 0006 Wounds that do not readily heal can cause the sub BACKGROUND OF THE INVENTION ject considerable physical, emotional, and social distress as 0003. The primary goal in the treatment of wounds is to well as great financial expense (Richey et al., 1989, Annals of achieve wound closure. Open cutaneous wounds represent Plastic Surgery 23:159). Indeed, wounds that fail to heal one major category of wounds and include burn wounds, properly and become infected may require excision of the wounds resulting from chemical (especially alkali) burns, affected tissue. A number of treatment modalities have been wounds from physical trauma, neuropathic ulcers, pressure developed as Scientists basic understanding of wounds and Sores, venous stasis ulcers, and diabetic ulcers. Open cutane wound healing mechanisms has progressed. ous wounds routinely heal by a process which comprises six 0007. The most commonly used conventional modality to major components: i) inflammation, ii) fibroblast prolifera assist in wound healing involves the use of wound dressings. tion, iii) blood vessel proliferation, iv) connective tissue syn In the 1960s, a major breakthrough in wound care occurred thesis, v) epithelialization, and vi) wound contraction. Wound when it was discovered that wound healing with moist, occlu healing is impaired when these components, either individu sive dressings was, generally speaking, more effective than ally or as a whole, do not function properly. Numerous factors the use of dry, non-occlusive dressings (Winter, 1962, Nature can affect wound healing, including but not limited to mal 193:293). Today, numerous types of dressings are routinely nutrition, systemic debility due to a variety of causes, wound used, including films (e.g., polyurethane films), hydrocol infection, local lack of progenitor cells, local and/or systemic loids (hydrophilic colloidal particles bound to polyurethane pharmacological agents (e.g., numerous chemotherapeutic foam), hydrogels (cross-linked polymers containing about at agents, actinomycin and steroids), repeated local trauma, dia least 60% water), foams (hydrophilic or hydrophobic), cal betes and other endocrine/metabolic diseases (e.g., Cushing's cium alginates (nonwoven composites of fibers from calcium disease), and advanced age (Hunt and Goodson, 1988, Cur alginate), and cellophane (cellulose with a plasticizer) (Kan rent Surgical Diagnosis & Treatment, Appleton & Lange, pp. non and Garrett, 1995, Dermatol. Surg. 21:583; Davies, 1983, 86-98). Additionally, wounds that are extensive in size, Burns 10:94). Unfortunately, certain types of wounds (e.g., regardless of the initiating cause, present special challenges diabetic ulcers, pressure Sores) and the wounds of certain due to the large surface area that must be re-epithelialized to Subjects (e.g., recipients of exogenous corticosteroids) do not re-establish Surface integrity. heal in a timely manner (or at all) with the use of such 0004 Delayed wound healing causes substantial morbid dressings. ity in subjects with diabetes. Diabetes mellitus is a chronic 0008. Several pharmaceutical modalities have also been disorder of glucose metabolism and homeostasis that dam utilized in an attempt to improve wound healing. For ages many organs. It is the eighth leading cause of death in the example, Some practitioners have utilized treatment regimens United States (Harris et al., 1987, Diabetes 36:523). In per involving zinc sulfate. However, the efficacy of these regi Sons with diabetes, vascular disease, neuropathy, infections, mens has been primarily attributed to their reversal of the and recurrent trauma predispose the extremities, especially effects of Sub-normal serum Zinc levels (e.g., decreased host the foot, to pathologic changes. These pathological changes resistance and altered intracellular bactericidal activity) (Ri can ultimately lead to chronic ulceration, which may neces ley, 1981, Am. Fam. Physician 24:107). While other vitamin sitate amputation. Chronic wounds and wounds with patho and mineral deficiencies have also been associated with logical or dysregulated healing represent a major health bur decreased wound healing (e.g., deficiencies of vitamins A, C den and drain on health care resources. Chronic wounds have and D; and calcium, magnesium, copper, and iron), there is no major impacts on the physical and mental health, productiv strong evidence that increasing the serum levels of these ity, morbidity, mortality and cost of care for affected individu Substances above their normal levels actually enhances als. The most common types of chronic wounds are caused by wound healing. Thus, except in very limited circumstances, systemic diseases such as diabetes, vascular problems such as the promotion of wound healing with these agents has met venous hypertension and by immobility-induced pressure with little success. sores; accounting for 70% of all chronic wounds. Statistics on 0009 Current clinical approaches used to promote healing the prevalence of chronic wounds varies, however studies in dysregulated wounds include protection of the wound bed report that 0.2% to 1% of the population suffer from venous from mechanical trauma (e.g. splinting, bandaging), meticu US 2011/O 1892.87 A1 Aug. 4, 2011
lous control of surface microbial burden (antibiotics, antimi reference in its entirety) is toxic to in vitro monolayer cell crobial peptides, bacteriophages, antiseptics and other anti cultures of keratinocytes and fibroblasts (Poon et al., 2004, microbial compounds that broadly inhibit wound pathogens Burns 30:140; Trop et al., 2006, J. Trauma 60:648; each (e.g., silver Sulfadiazine) combined with topical application herein incorporated by reference in its entirety). of soluble cytoactive factors (e.g. growth factors exemplified 0013 The complex nature of pathologic wounds, and the by but not limited to epidermal growth factor-EGF, exog lack of significant clinical progress based on current thera enous extracellular matrix constituents such as fibronectin), pies, indicates the urgent need for new and unconventional Surgical excision of the wound margin or entire bed and approaches. What is needed is are safe, effective, and inter Surgical placement of tissue flaps and/or autografts, allografts active means for enhancing the healing of chronic and severe and Xenografts. All of these approaches fall short of promot wounds. The methods should be adaptable without regard to ing optimal healing conditions in many of the most challeng the type of wound, or the nature of the patient population, to ing wounds. It is likely that a major contributing factor to the which the subject belongs. failure of these traditional approaches is the fact that they do not alter the intrinsic chemistry/structure of the wound bed SUMMARY OF THE INVENTION itself that has been shown in many cases to contribute signifi 0014. The present invention relates to methods and com cantly to their persistence. Additionally, the historical use of positions for the modulation of wound healing. In particular, a single factor or set of factors to treat all wounds often falls the present invention relates to promoting and enhancing short due to the great heterogeneity found in wound beds wound healing by changing the intrinsic chemical composi themselves and the complex environment of the wound itself tion and/or physical attributes of the wound bed. Accordingly, containing a community of signaling molecules that fre in some embodiments the present invention provides formu quently modulate the activity of individual molecules. lations that alter the physical attributes (compliance, topog 0010. Of broad-spectrum bactericidal agents, silver is raphy, charge), as well as cross-linker covalent modification considered particularly favorable because the likelihood of molecules to attach and deliver antimicrobial compounds as developing bacterial resistance to silver is believed to be very well as extracellular matrices and other scaffolds and cytoac low; therefore, it can be employed as a bactericidal agent tive agents to a wound. In addition, the present invention continuously. However, currently available methods of provides methods and compositions utilizing oppositely applying silver as a bactericidal agent for wound treatment charged polyelectrolytes to form a polyelectrolyte layer on a are inadequate. For example, 0.5% silver nitrate solution is a wound surface. The invention further relates to the incorpo standard and popular agent for topical burn wound therapy, ration of non-charged polymers into the wound bed via inter providing a beneficial effect in decreasing wound Surface actions such as but not limited to hydrogen bonding. The inflammation. However, while such formulations have a high Scope of the invention includes incorporation of nanoparticles concentration of silver, there is no residual activity, necessi and microparticles into the wound bed to engineer the physi tating frequent applications (e.g., up to 12 times a day) which cal characteristics and chemical composition of the wound poses a severe logistical burden in clinical settings. Silver bed. The invention further relates to incorporating wound ions released through use of 0.5% silver nitrate solution active agents into a polyelectrolyte layer, nanoparticle or become rapidly inactive through formation of chemical com microparticle for delivery to a wound. In some embodiments, plexes by chloride within 2 hours. Frequent dressings also the present invention provides formulations that promote a result in large excesses of silver being delivered to the wound, favorable and stable pH, Surface charge, Surface energy, causing wound-discoloration and toxic effects (Dunn et al., osmotic environment, Surface functionalities that enhance 2004, Burns 30(supplement 1):S1; herein incorporated by galvano and magneto positive effects on wound healing, Sup reference in its entirety). Additionally, nitrate is toxic to ply of nitric oxide, provide energy sources for cells and/or wounds and to mammalian cells. The reduction of nitrate to provide a balance of MMP/other peptidase/protease activity. nitrite further causes oxidant-induced damage to cells, which 0015 The present invention further relates to providing is cited as the most likely reason for the impaired re-epithe compositions, methods, and kits comprising selectively toxic lialization with use of silver nitrate solution in partial thick wound active agents that are highly bactericidal but that Sup ness burns or donor sites. port growth and viability of mammalian cells (e.g., kerati 0011 Silver compounds such as silver sulfadiazine in nocytes, neurons, vascular endothelial cells and fibroblasts cream formulations (e.g., Flammazine R, SilvadeneR) have cells). In preferred embodiments, the wound active agent is also been used for wound treatment. However, such formu silver, including but not limited to silver nanoparticles. In lations also have limited residual activity and have to be particularly preferred embodiments, the silver loading of applied twice a day. Bacterial resistance does develop to these some embodiments of the present invention is between 0.35 formulations, and, impaired re-epithelialization has also been and 0.4 g/cm. Polyelectrolyte thin films of the present observed. Bone marrow toxicity has been observed with sil invention comprising silver (e.g., silver nanoparticles) find ver Sulfadiazine, primarily due to its propylene glycol com use in the treatment of wounds and the prevention of infec ponent. tion, including but not limited to as coatings on devices that 0012. Additionally, in some methods, silver itself is incor come partially, directly, indirectly, or completely in contact porated into the dressing instead of being applied as a separate with the body of a Subject (e.g., a human patient). formulation. Controlled and prolonged release of silver to the 0016. In one embodiment, the compositions and methods wound allows dressings to be changed less frequently. How of the present invention provide approaches to promote heal ever, dressings have to be impregnated with large amount of ing in pathologic wounds by altering the Surface chemistry silver, which results in cytotoxicity to mammalian cells. Sil and structure of the pathologic wound bed itself so as to ver released from a commercially available wound-dressing ultimately enable differential modulation of key cellular ele (ActicoatTM) containing nanocrystalline silver (Dunn et al., ments customizable to the specific patient's health wound 2004, Burns 30(supplement 1):S1; herein incorporated by type and anatomic location in a Subject. US 2011/O 1892.87 A1 Aug. 4, 2011
0017. In one embodiment, the present invention provides trophic factors, extracellular matrices, enzymes, enzyme for the covalent immobilization of factors to the wound bed. inhibitors, defensins, polypeptides, anti-infective agents, In some embodiments, the present invention provides meth buffering agents, vitamins and minerals, analgesics, antico ods of treatment, comprising providing a Subject having a agulants, coagulation factors, anti-inflammatory agents, wound, at least one covalent modification agent and at least vasoconstrictors, vasodilators, diuretics, and anti-cancer one wound active agent, and contacting the wound with the at agents. In some embodiments, the at least one wound active least one covalent modification agent and the at least one agent contains one or more free -SH groups. wound active agent under conditions such that the at least one 0018. The present invention also provides a kit for treating wound active agent is covalently attached to the wound. In a subject having a wound, comprising at least one covalent Some embodiments, the Subject is a human. In other embodi modification agent, at least one wound active agent, and ments, the Subject is a non-human vertebrate. In some instructions for using the kit to covalently link the at least one embodiments, the at least one covalent modification agent is wound active agent to the wound. In some embodiments, the a homobifunctional cross-linker. In other embodiments, theat at least one covalent modification agentis a homobifunctional least one covalent modification agent is a heterobifunctional cross-linker. In some embodiments, the homobifunctional cross-linker. For example, in some embodiments, the homo cross-linker is an N-hydroxySuccinimidyl ester (e.g., includ bifunctional cross-linker is an N-hydroxysuccinimidyl ester ing, but not limited to, disuccinimidyl ester, dithiobis(Succin (e.g., including, but not limited to, disuccinimidyl ester, imidylpropionate), 3,3'-dithiobis(sulfosuccinimidylpropi dithiobis(succinimidylpropionate), 3,3'-dithiobis(sulfosuc onate), disuccinimidyl Suberate, bis(SulfoSuccinimidyl) cinimidylpropionate), disuccinimidyl Suberate, bis(Sulfosuc Suberate, disuccinimidyl tartarate, disulfo Succinimidyl cinimidyl)Suberate, disuccinimidyl tartarate, disulfoSuccin tartarate, bis 2-(Succinimidyloxycarbonyloxy)ethylsulfone, imidyl tartarate, bis2-(Succinimidyloxycarbonyloxy)ethyl bis(2-(sulfosuccinimidooxycarbonyloxy)ethylsulfone, eth Sulfone, bis(2-(sulfosuccinimidooxycarbonyloxy)ethyl ylene glycolbis(Succinimidylsuccinate), ethylene glycolbis Sulfone, ethylene glycolbis(Succinimidylsuccinate), ethylene (sulfoSuccinimidylsuccinate), disuccinimidyl glutarate, and glycolbis(SulfoSuccinimidylsuccinate), disuccinimidyl glut N,N'-disuccinimidylcarbonate). In some embodiments, the at arate, and N,N'-disuccinimidylcarbonate). In some embodi least one covalent modification agent is a heterobifunctional ments, the homobifunctional cross-linker is at a concentra cross-linker (e.g., including, but not limited to, N-Succinim tion between 1 nanomolar and 10 millimolar. In some idyl 3-(2-pyridyldithio)propionate, succinimidyl 6-(3-2-py preferred embodiments, the homobifunctional cross-linker is ridyldithio-propionamido)hexanoate, SulfoSuccinimidyl at a concentration between 10 micromolar and 1 millimolar. 6-(3'-(2-pyridyldithio-propionamido)hexanoate, succinim In other embodiments, the at least one covalent modification idyloxycarbonyl-O-methyl-O-(2-pyridyldithio)toluene, Sul agent is a heterobifunctional cross-linker (e.g., including, but fosuccinimidyl-6-O-methyl-O-(2-pyridyldithio)toluamido not limited to, N-succinimidyl 3-(2-pyridyldithio)propi hexanoate, Succinimidyl 4-(N-maleimidomethyl) onate. Succinimidyl 6-(3-2-pyridyldithio-propionamido) cyclohexane-1-carboxylate, SulfoSuccinimidyl 4-(N- hexanoate, sulfosuccinimidyl 6-(3'-2-pyridyldithio-propi maleimidomethyl)cyclohexane-1-carboxylate, onamido)hexanoate, Succinimidyloxycarbonyl-O-methyl-O- m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-male (2-pyridyldithio)toluene, sulfosuccinimidyl-6-O-methyl-O- imidobenzoyl-N-hydroxy-sulfosuccinimide ester, N-succin (2-pyridyldithio)toluamidohexanoate, succinimidyl 4-(N- imidyl(4-iodoacetyl)aminobenzoate, Sulfo-Succinimidyl(4- maleimidomethyl)cyclohexane-1-carboxylate, iodoacetyl)aminobenzoate, Succinimidyl-4-(p- sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p- carboxylate, m-maleimidobenzoyl-N-hydroxySuccinimide maleimidophenyl)butyrate, N-(y-maleimidobutyryloxy) ester, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide Succinimide ester, N-(y-maleimidobutyryloxy) ester, N-Succinimidyl(4-iodoacetyl)aminobenzoate, Sulfo SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) Succinimidyl(4-iodoacetyl)aminobenzoate, Succinimidyl-4- hexanoate, Succinimidyl 6-(6-(((4-iodoacetyl)amino) (p-maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p-male hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) imidophenyl)butyrate, N-(y-maleimidobutyryloxy) amino)methyl)cyclohexane-1-carboxylate, Succinimidyl Succinimide ester, N-(y-maleimidobutyryloxy) 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) amino)-hexanoate, and p-nitrophenyl iodoacetate). In some hexanoate, succinimidyl 6-(6-(((4-iodoacetyl)amino) embodiments, the at least one wound active agent includes, hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) but is not limited to, trophic factors (including polypetide amino)methyl)cyclohexane-1-carboxylate, Succinimidyl growth factors, neuropeptides, neurotrophins, extracellular 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) matrices and their individual native constituents (exemplified amino)-hexanoate, and p-nitrophenyl iodoacetate). In some by but not limited to laminin, fibronectin, vitronectin, col embodiments, the heterobifunctional cross-linker is modified lagens, also select amino acid sequences found in these pro with functional groups, rendering it soluble in aqueous sol teins known to promote cell behaviors favorable to wound vents for delivery as an aqueous solution. Furthermore, in healing e.g., integrin binding sequences exemplified by but Some embodiments, the aqueous solution contains additives not limited to RGD, EILDV,VCAM-1 and their recombined (e.g., including, but not limited to, Surfactants and block or synthetic analogs, enzymes, enzyme inhibitors, polypep copolymers). In other embodiments, a multiplicity of hetero tides, antimicrobial peptides (exemplified by but not limited bifunctional cross-linkers can be attached to a molecule, to defensins, magaignins, cathelocidins, bactenicin) anti-in polymer or particle to serve as the cross-linking agent. In fective agents including silver containing compounds (e.g., other embodiments, the heterobifunctional cross-linker is ionic silver, elemental silver, silver nanoparticles, and formu dissolved in an organic solvent (e.g., including, but not lim lations thereof), buffering agents, vitamins and minerals, ited to, dimethyl sulfoxide). In some embodiments, the at compounds that promote generation/stabilization of nitic least one wound active agent includes, but is not limited to, oxide, energy sources for cells, analgesics, anticoagulants, US 2011/O 1892.87 A1 Aug. 4, 2011 coagulation factors, anti-inflammatory agents, vasoconstric amino)methyl)cyclohexane-1-carboxylate, Succinimidyl tors, vasodilators, diuretics, and anti-cancer agents. In other 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) embodiments the kits include small interfering RNAs (siR amino)-hexanoate, and p-nitrophenyl iodoacetate). In some NAS-also referred to as micro RNAs) that are capable of embodiments, the at least one covalent modification agent is promoting cellular behaviors conducive to the wound healing a homobifunctional cross-linker In some embodiments, the process. In other embodiments, the kits include compounds homobifunctional cross-linker is an N-hydroxysuccinimidyl that promote/stabilize a favorable pH, osmotic environment, ester (e.g., including, but not limited to, disuccinimidyl ester, Surface energy, Surface charge, Surface functionalities that dithiobis(succinimidylpropionate), 3,3'-dithiobis(sulfosuc enhance galvano and magneto positive effects on woundheal cinimidylpropionate), disuccinimidyl Suberate, bis(Sulfo Suc ing, or balance of MMP/other peptidase/protease activity. cinimidyl)Suberate, disuccinimidyl tartarate, disulfoSuccin 0019. In some embodiments, the present invention pro imidyl tartarate, bis2-(Succinimidyloxycarbonyloxy)ethyl vides a method of treatment, comprising providing a subject Sulfone, bis 2-(sulfoSuccinimidooxycarbonyloxy)ethyl having a wound, at least one cationic polyelectrolyte, at least Sulfone, ethylene glycolbis(Succinimidylsuccinate), ethylene one anionic polyelectrolyte, at least one covalent modifica glycolbis(SulfoSuccinimidylsuccinate), disuccinimidyl glut tion agent, and at least one wound active agent, and contacting arate, and N,N'-disuccinimidylcarbonate). In some embodi the wound with the at least one cationic and the at least one ments, the at least one covalent modification agent can be at a anionic polyelectrolytes, the at least one covalent modifica concentration between 1 nanomolar and 10 millimolar. In tion agent, and the at least one wound active agent so that the Some preferred embodiments, the at least one covalent modi at least one wound active agent is covalently linked to the fication agent is at a concentration between 10 micromolar to wound by incorporation into a polyelectrolyte layer formed 1 millimolar. In some preferred embodiments, the at least one by the at least one cationic and the at least one anionic poly covalent modification agent is bis(SulfoSuccinimidyl)Suber electrolytes. In some preferred embodiments, the polyelec ate (for example, in aqueous Solution at a concentration trolytes are sequentially and repeatedly layered on the wound, between 1 nM and 10 mM). In some embodiments, the at least then the at least one covalent modification agent and the at one covalent modification agent comprises N-hydroxysuc least one wound active agent are added. In some embodi cinimidyl ester. In some preferred embodiments, the at least ments, the at least one cationic polyelectrolyte (e.g., includ one wound active agent contains the peptide sequence gly ing, but not limited to, poly(L-lysine), poly(ethylene imine), arg-gly-asp-ser-pro-lys. and poly(allylamine hydrochloride and dendrimers and 0020. In some embodiments, a first at least one anionic or multi-armed polymers that present multiple amine groups) is cationic polyelectrolyte is contacted with the at least one the top layer of the polyelectrolyte layers. Furthermore, in covalent modification agent and the at least one wound active Some embodiments, the at least one cationic polyelectrolyte agent before contacting with the wound bed and oppositely harbors a primary amine group which allows attachment of charged at least one cationic or anionic polyelectrolyte. In the at least one covalent modification agent to the at least one Some preferred embodiments, the first at least one anionic cationic polyelectrolyte. For example, in some preferred polyelectrolyte is polyglutamic acid, the at least one covalent embodiments, the at least one cationic polyelectrolyte is modification agentis N-hydroxysuccinimidyl ester, and theat polylysine and the at least one anionic polyelectrolyte is least one wound active agent contains a primary amine (e.g., polyglutamic acid. In some embodiments, the at least one including, but not limited to, the peptide sequence gly-arg anionic polyelectrolyte (e.g., including, but not limited to, gly-asp-ser-pro-lys). In some preferred embodiments, the poly(L-glutamic acid), poly(sodium 4-styrenesulfonate), first at least one cationic polyelectrolyte is polylysine, the at poly(acrylic acid), poly(maleic acid-co-propylene), hyalu least one covalent modification agentis N-Succinimidyl 3-(2- ronic acid, chondroitin, and poly(vinyl sulfate)) is the top pyridyldithio)propionate, and the at least one wound active layer of the polyelectrolyte layers. In a preferred embodi agent contains the peptide sequence gly-arg-gly-asp-ser-pro ment, the at least one anionic polyelectrolyte is polyglutamic cyS. acid. In some embodiments, the at least one covalent modi 0021. The present invention further provides kits for treat fication agent is a heterobifunctional cross-linker (e.g., inga Subject having a wound, comprising at least one cationic including, but not limited to, N-Succinimidyl 3-(2-py polyelectrolyte, at least one anionic polyelectrolyte, at least ridyldithio)propionate, succinimidyl 6-(3-2-pyridyldithio one covalent modification agent, at least one wound active propionamido)hexanoate, SulfoSuccinimidyl 6-(3'-2-py agent; and instructions for using the kit to covalently link the ridyldithio-propionamido)hexanoate, at least one wound active agent to the wound by incorporation Succinimidyloxycarbonyl-O-methyl-O-(2-pyridyldithio) into a polyelectrolyte layer that is formed by the at least one toluene, sulfosuccinimidyl-64C.-methyl-O-(2-pyridyldithio) cationic and anionic polyelectrolytes. In some embodiments, toluamidohexanoate, Succinimidyl 4-(N-maleimidomethyl) the incorporation of the at least one wound active agent is cyclohexane-1-carboxylate, SulfoSuccinimidyl 4-(N- achieved by sequential and repeated layering of the polyelec maleimidomethyl)cyclohexane-1-carboxylate, trolytes, followed by addition of the at least one covalent m-maleimidobenzoyl-N-hydroxySuccinimide ester, m-male modification agent and the at least one wound active agent. In imidobenzoyl-N-hydroxy-sulfosuccinimide ester, N-succin other embodiments, a first at least one anionic or cationic imidyl(4-iodoacetyl)aminobenzoate, Sulfo-Succinimidyl(4- polyelectrolyte is contacted with the at least one covalent iodoacetyl)aminobenzoate, Succinimidyl-4-(p- modification agent and the at least one wound active agent maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p- before contacting with the woundbed and oppositely charged maleimidophenyl)butyrate, N-(y-maleimidobutyryloxy) at least one cationic or anionic polyelectrolyte. In some Succinimide ester, N-(y-maleimidobutyryloxy) embodiments, the at least one cationic polyelectrolyte SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) includes, but is not limited to, poly(L-lysine), poly(ethylene hexanoate, succinimidyl 6-(6-(((4-iodoacetyl)amino) imine), and poly(allylamine hydrochloride). In some embodi hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) ments, the at least one anionic polyelectrolyte includes, but is US 2011/O 1892.87 A1 Aug. 4, 2011
not limited to, poly(L-glutamic acid), poly(sodium 4-styre ing but not limited to an anti-infective agent (e.g., ionic silver, nesulfonate), poly(acrylic acid), poly(maleic acid-co-propy elemental silver, silver nanoparticles, and formulations lene), and poly(vinyl Sulfate). In some embodiments, the at thereof) least one covalent modification agent is a homobifunctional 0023 The present invention further provides a kit for treat cross-linker. In some embodiments, the homobifunctional ing a subject having a wound, comprising: at least one cat cross-linker is an N-hydroxysuccinimidyl ester (e.g., includ ionic polyelectrolyte, at least one anionic polyelectrolyte, at ing, but not limited to, disuccinimidyl ester, dithiobis(Succin least one DNA delivery agent, at least one DNA species, and imidylpropionate), 3,3'-dithiobis(sulfosuccinimidylpropi instructions for using the kit to deliver the at least one DNA onate), disuccinimidyl Suberate, bis(SulfoSuccinimidyl) species to the wound using the at least one DNA delivery Suberate, disuccinimidyl tartarate, disulfo Succinimidyl agent and a polyelectrolyte layer formed by the at least one tartarate, bis 2-(Succinimidyloxycarbonyloxy)ethylsulfone, cationic and anionic polyelectrolytes. In some embodiments, bis(2-(sulfosuccinimidooxycarbonyloxy)ethylsulfone, eth the kit further comprises an additional wound active agent ylene glycolbis(Succinimidylsuccinate), ethylene glycolbis (including but not limited to an anti-infective agent (e.g., (SulfoSuccinimidylsuccinate), disuccinimidyl glutarate, and ionic silver, elemental silver, silver nanoparticles, and formu N,N'-disuccinimidylcarbonate). In other embodiments, the at lations thereof) least one covalent modification agent is a heterobifunctional 0024. The present invention also provides a method of cross-linker (e.g., including, but not limited to, N-Succinim treatment, comprising providing a Subject having a wound, a idyl 3-(2-pyridyldithio)propionate, succinimidyl 6-(3-2-py cationic and anionic polyelectrolyte mixture, a deprotection ridyldithio-propionamido)hexanoate, SulfoSuccinimidyl agent, at least one covalent modification agent, and at least one wound active agent; contacting the wound with the poly 6-(3'-2-pyridyldithio-propionamido)hexanoate. Succinim electrolyte mixture to form a polyelectrolyte layer on the idyloxycarbonyl-O-methyl-O-(2-pyridyldithio)toluene, Sul wound; applying the deprotection agent to the polyelectrolyte fosuccinimidyl-6-O-methyl-O-(2-pyridyldithio)toluamido layer to form a deprotected polyelectrolyte layer, applying hexanoate, Succinimidyl 4-(N-maleimidomethyl) the at least one covalent modification agent to the deprotected cyclohexane-1-carboxylate, SulfoSuccinimidyl 4-(N- polyelectrolyte layer to form a modified deprotected poly maleimidomethyl)cyclohexane-1-carboxylate, electrolyte layer, and applying the at least one wound active m-maleimidobenzoyl-N-hydroxySuccinimide ester, m-male agent to the modified deprotected polyelectrolyte layer under imidobenzoyl-N-hydroxy-sulfosuccinimide ester, N-succin conditions such that the at least one wound active agent is imidyl(4-iodoacetyl)aminobenzoate, sulfo-succinimidyl (4- covalently attached to the wound. In some preferred embodi iodoacetyl)aminobenzoate, Succinimidyl-4-(p- ments, the anionic and cationic polyelectrolyte mixture is maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p- made up of polylactic acid and poly(epsilon-CBZ-L-lysine), maleimidophenyl)butyrate, N-(y-maleimidobutyryloxy) blended at an 80:20 ratio. In some embodiments, the depro Succinimide ester, N-(y-maleimidobutyryloxy) tection occurs by acid hydrolysis. In some embodiments, the SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) at least one covalent modification agent is SP3. The present hexanoate, succinimidyl 6-(6-(((4-iodoacetyl)amino) invention also provides a composition comprising a depro hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) tected polyelectrolyte, functionalized with at least one cova amino)methyl)cyclohexane-1-carboxylate, Succinimidyl lent modification agent containing an active group that is 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) exposable to at least one wound active agent. In some pre amino)-hexanoate, and p-nitrophenyl iodoacetate). In some ferred embodiments, the deprotected polyelectrolyte is com embodiments, the at least one wound active agent includes, prised of polylactic acid and poly(epsilon-CBZ-L-lysine), but is not limited to, trophic factors, extracellular matrices, blended at an 80:20 ratio. In a preferred embodiment, the at enzymes, enzyme inhibitors, defensins, polypeptides, anti least one covalent modification agent is SP3. 0025. The present invention also provides a method of infective agents (including but not limited to silver (e.g., ionic treatment, comprising providing a Subject having a wound, a silver, elemental silver, silver nanoparticles, and formulations cationic and anionic polyelectrolyte mixture, a deprotection thereof)), buffering agents, vitamins and minerals, analge agent, at least one covalent modification agent, a first sics, anticoagulants, coagulation factors, anti-inflammatory polypeptide, and at least one wound active agent linked to a agents, vasoconstrictors, vasodilators, diuretics, and anti second polypeptide; contacting the wound with the polyelec cancer agents. trolyte mixture to form a polyelectrolyte layer on the wound; 0022. The present invention also provides a method of applying the deprotection agent to the polyelectrolyte layer to treatment, comprising providing a Subject having a wound, at form a deprotected polyelectrolyte layer, applying the at least least one cationic polyelectrolyte, at least one anionic poly one covalent modification agent to the deprotected polyelec electrolyte, at least one DNA delivery agent, and at least one trolyte layer to form a modified deprotected polyelectrolyte DNA species, and contacting the wound with the at least one layer; applying the first polypeptide to the modified depro cationic polyelectrolyte, the at least one anionic polyelectro tected polyelectrolyte layer to covalently link the first lyte, the at least one DNA delivery agent, and the at least one polypeptide to the modified deprotected polyelectrolyte DNA species under conditions such that the at least one DNA layer; and applying the at least one wound active agent linked species is delivered to the wound. In some preferred embodi to the second polypeptide to the modified deprotected poly ments, the at least one DNA species includes, but is not electrolyte layer covalently linked to the first polypeptide, limited to, DNA encoding vascular endothelial growth factor under conditions such that the at least one wound active agent and/or epidermal growth factor. In some preferred embodi is attached to the wound by specific protein binding between ments, the at least one cationic polyelectrolyte is polylysine. the first polypeptide and the second polypeptide. In some In Some embodiments, the method further comprises contact preferred embodiments, the specific protein binding occurs ing the wound with an additional wound active agent (includ between biotin and a polypeptide (e.g., including, but not US 2011/O 1892.87 A1 Aug. 4, 2011
limited to, avidin, neutravidin, and Streptavidin). In other Some preferred embodiments, the at least one covalent modi preferred embodiments, the specific protein binding occurs fication agent is SP3. In some embodiments, the kit also between glutathione-S-transferase and glutathione. In yet contains a first and a second polypeptide that interact with other preferred embodiments, the specific protein binding each other by specific protein binding. In some preferred occurs between nickel-nitrilotriacetic acid and polyhistidine. embodiments, the first polypeptide is linked to either the at In some embodiments, the at least one covalent modification least one covalent modification agent or the at least one agent is SP3. wound active agent and the second polypeptide is linked to 0026. The present invention further provides a composi the other at least one covalent modification agent or the at tion comprising a deprotected polyelectrolyte functionalized least one wound active agent. In some preferred embodi with at least one covalent modification agent linked to a first ments, the first polypeptide is biotin and the second polypep polypeptide that interacts by specific binding with a second tide is avidin, neutravidin, or streptavidin. In some other polypeptide linked to at least one wound active agent. In some preferred embodiments, the first polypeptide is glutathione preferred embodiments, the specific binding occurs between S-transferase and the second polypeptide is glutathione. In biotin and a polypeptide (e.g., including, but not limited to, still other preferred embodiments, the first polypeptide is avidin, neutravidin, and streptavidin). In other preferred nickel-nitrilotriacetic acid and the second polypeptide is embodiments, the specific binding occurs between glu polyhistidine. In some embodiments, the kit contains at least tathione-S-transferase and glutathione. In still other preferred one molecule containing an azide group that is attached to the embodiments, the specific protein binding occurs between polyelectrolyte mixture after deprotection so that at least one nickel-nitrilotriacetic acid and polyhistidine. wound active agent containing an alkyne group can be 0027. The present invention also provides a method of attached to the wound by click chemistry. treatment, comprising providing a Subject having a wound, a 0030 The present invention further provides a method of cationic and anionic polyelectrolyte mixture, a deprotection treatment, comprising providing a Subject having a wound, at agent, at least one covalent modification agent, at least one least one cationic polyelectrolyte, at least one anionic poly molecule containing an azide group, and at least one wound electrolyte, at least one modifying agent, and at least one active agent containing an alkyne group; contacting the wound active agent containing an amino terminal cysteine wound with the polyelectrolyte mixture to form a polyelec residue; and contacting the wound with the polyelectrolytes, trolyte layer on the wound; applying the deprotection agent to the at least one modifying agent and the at least one wound the polyelectrolyte layer to form a deprotected polyelectro active agent so that the at least one wound active agent is lyte layer; applying the at least one covalent modification attached to the wound by native chemical ligation. In some agent to the deprotected polyelectrolyte layer to form a modi preferred embodiments, the polyelectrolytes are sequentially fied deprotected polyelectrolyte layer, applying the at least and repeatedly layered on the wound. In some embodiments, one molecule containing an azide group to the modified the at least one anionic polyelectrolyte (e.g., including, but deprotected polyelectrolyte layer to covalently link the at not limited to, polyglutamic acid) is the top layer of the least one molecule containing an azide group to the modified polyelectrolyte layers. In some embodiments, the at least one deprotected polyelectrolyte layer, and applying the at least modifying agents are ethylene dichloride and HSCHPh. one wound active agent containing an alkyne group to the 0031. The present invention also provides a composition modified deprotected polyelectrolyte layer covalently linked comprising a polyelectrolyte layer that is functionalized with to the at least one molecule containing an azide group, so that at least one modifying agent that is exposable to native chemi the at least one wound active agent is attached to the wound by calligation with at least one wound active agent containing an click chemistry. In some preferred embodiments, the at least amino terminal cysteine residue. one molecule containing an azide group has the formula 0032. The present invention also provides a kit for treating HN(CH2CH2O)N. In some preferred embodiments, the at a Subject having a wound, comprising at least one cationic least one wound active agent is L-propargylglycine. In some polyelectrolyte, at least one anionic polyelectrolyte, at least preferred embodiments, the reaction is carried out in the one modifying agent, at least one wound active agent con presence of Cu(I). taining an amino terminal cysteine residue, and instructions 0028. The present invention further provides a composi for using the kit to form a polyelectrolyte layer on the wound, tion comprising a deprotected polyelectrolyte functionalized followed by treatment with the at least one modifying agent with at least one covalent modification agent linked to at least and attachment of the at least one wound active agent by one molecule containing an azide group that is attached by native chemical ligation. In some preferred embodiments, the click chemistry to at least one wound active agent containing at least one anionic polyelectrolyte is polyglutamic acid. In an alkyne group. Some preferred embodiments, the at least one modifying 0029. The present invention further provides a kit for treat agents are ethylene dichloride and HSCHPh. ing a subject having a wound, comprising a cationic and 0033. In some embodiments, the present invention pro anionic polyelectrolyte mixture, a deprotection agent, at least vides methods of treatment comprising: a) providing a Sub one covalent modification agent, at least one wound active ject having a wound, at least one wound modifying agent, and agent, and instructions for using the kit to covalently link the at least one wound active agent; b) contacting the wound with at least one wound active agent to the wound by deprotection the at least one wound modification agent to provide a modi of the polyelectrolyte mixture contacted to the wound, fol fied wound bed, and c) contacting the modified wound bed lowed by addition of the at least one covalent modification with the at least one wound active agent underconditions such agent and the at least one wound active agent. In some pre that the at least one wound active agent is incorporated into ferred embodiments, the polyelectrolyte mixture comprises the modified wound bed and healing of the wound is polylactic acid and poly(epsilon-CBZ-L-lysine). In some enhanced. In some embodiments, the modified wound bed is preferred embodiments, the deprotection agent causes depro modified to present a functionalized surface reactive with the tection by acid hydrolysis of the polyelectrolyte mixture. In at least one wound active agent. In some embodiments, the US 2011/O 1892.87 A1 Aug. 4, 2011
wound active agent is applied to the modified wound bed to azide group or alkyne group, and V) at least one wound active form a gradient. In some embodiments, the wound modifying agent containing the other of an azide group or an alkyne agent alters a property of the wound bed selected from the group; b) contacting the wound with the polyelectrolyte mix group consisting of compliance, pH, alkalinity, and oxidative ture to form a polyelectrolyte layer on the wound; c) applying or reductive strength, net charge, hydrophilicity, osmotic the deprotection agent to the polyelectrolyte layer to form a strength, nanoscale or Submicron topographic features, elec deprotected polyelectrolyte layer; d) applying the at least one troconductivity, MMP production, phagocytosis, and trans covalent modification agent to the deprotected polyelectro glutaminase activity. In some embodiments, the wound modi lyte layer to form a modified deprotected polyelectrolyte fying agent is applied to the woundbed by a method selected layer; e) applying the at least one molecule containing an from the group consisting of coating, sprinkling, seeding, azide group or an alkyne group to the modified deprotected electrospinning, spattering, stamping, spraying, pumping, polyelectrolyte layer to covalently link the molecule contain painting, Smearing and printing. In some embodiments, the at ing an azide or an alkyne group to the modified deprotected least one wound modification agent is at least one crosslinker. polyelectrolyte layer, f) applying the at least one wound In some embodiments, the at least one crosslinker is selected active agent containing the other of an azide group or an from the group consisting of a homobifunctional crosslinker, alkyne group to the modified deprotected polyelectrolyte a heterobifunctional cross linker, a hetero- and homo-multi layer covalently linked to the molecule containing an azide functional crosslinker, and a photoactivatable crosslinker and group, under conditions such that the at least one wound combinations thereof. In some embodiments, the heterobi active agent is attached to the wound by click chemistry. functional crosslinker comprises an alkyne group. In some 0036. In some embodiments, the present invention pro embodiments, the wound modifying agent comprises at least vides a composition comprising a deprotected polyelectro one polymer. In some embodiments, the at least one polymer lyte functionalized with at least one covalent modification is applied to the wound bed to form a polymer multilayer. In agent linked to at least one molecule containing an azide Some embodiments, the at least one polymer is selected from group that is attached by click chemistry to at least one wound the group consisting of a cationic polymer, an anionic poly active agent containing an alkyne group. mer, a nonionic polymer an amphoteric polymer and combi 0037. In some embodiments, the present invention pro nations thereof. In some embodiments, the methods further vides methods comprising a) providing a subject having a comprise contacting the polymer multilayer with a wound, a plurality of functionalized beads and at least one crosslinker to form a functionalized surface reactive with the cytoactive factor, b) contacting the at least one cytoactive at least one wound active agent. In some embodiments, the at factor with the at least one functionalized biocompatible bead least one polymer is a polyelectrolyte multilayer preformed such that the at least one cytoactive factor is linked to the at on a Support so that the polyelectrolyte multilayer can be least one functionalized biocompatible bead and c) applying transferred from the support to the wound bed to form a the at least one functionalized biocompatible bead linked to modified woundbed. In some embodiments, the Support is an the at least one cytoactive factor to a woundbed of the subject. elastomeric Support. In some embodiments, the polymer mul 0038. In some embodiments, the present invention pro tilayer is from 1 nm to 250 nm thick. In some embodiments, vides methods of treatment comprising: a) providing a Sub the polymer multilayer has a compliance of from 3 to 500 kPa. ject having a wound, at least one polymer, and at least one In some embodiments, the wound modifying agentis selected wound active agent b) applying the at least one polymer to the from the group consisting of nanoparticles and micropar wound so that a polymer multilayer is formed on the wound; ticles. In some embodiments, the nano- and microparticles and c) incorporating the at least one wound active agent into are functionalized. In some embodiments, the functionalized the polymer multilayer during application of the at least one bead is a mesoscopic cross-linker. In some embodiments, the polymer, wherein the at least one wound active agent forms a at least one wound active agent is selected from the group gradient in the polymer multilayer. consisting of trophic factors, extracellular matrices, enzymes, 0039. In some embodiments, the present invention pro enzyme inhibitors, defensins, polypeptides, anti-infective vides an article comprising a matrix formed from a biocom agents, buffering agents, vitamins and minerals, analgesics, patible material, the matrix comprising at least one wound anticoagulants, coagulation factors, anti-inflammatory active agent, wherein the matrix is from 1 to 500 nm in agents, vasoconstrictors, vasodilators, diuretics, and anti thickness and has a compliance of from 3 to 500 kPa. In some cancer agents. embodiments, the matrix is functionalized. In some embodi 0034. In some embodiments, the present invention pro ments, the biocompatible material is selected from the group vides kits for treating a Subject having a wound, comprising: consisting of proteins and polymers. In some embodiments, a) at least one wound modification agent, b) at least one the polymers are selected from the group consisting of polya wound active agent; and c) instructions for using the kit to nionic polymers, polycationic polymers, uncharged poly treat a wound bed with the at least one wound modification mers, and amphoteric polymers and combinations thereof, agent to provide a modified wound bed and for incorporating and wherein the polymers from a multilayer. In some embodi the wound active agent into the modified wound bed. In some ments, the proteins are extracellular matrix proteins selected embodiments, the at least one wound modification agent is from the group consisting of laminin, vitronectin, fibronec selected from the group consisting of a covalent modifying tion, keratin, collagen, and combinations thereof. In some agent, at least one polyelectrolyte, microparticle, nanopar embodiments, the matrix is Supported by a solid Support ticle, and combinations thereof selected from the group consisting of silicone, siloxane elas 0035. In some embodiments, the present invention pro tomers, latex, nylon, nylon mesh, biological tissue, silk, poly vides methods of treatment comprising: a) providing a Sub urethane, Teflon, polyvinyl alcohol membranes, and polyeth ject having a wound, i) a cationic and anionic polyelectrolyte ylene oxide membranes. In some embodiments, the at least mixture, ii)a deprotection agent, iii) at least one covalent one wound active agent is distributed on the matrix so that a modification agent, iv) at least one molecule containing an gradient is formed. In some embodiments, the matrix is at US 2011/O 1892.87 A1 Aug. 4, 2011
least partially PEGylated. In some embodiments, the present the composition. In some embodiments, the wound active invention provides methods comprising, applying the articles agent is selected from the group consisting of antimicrobials, described above to a wound on a subject, wherein the article trophic factors, extracellular matrices, enzymes, enzyme enhances healing of the wound. In some embodiments, the inhibitors, defensins, polypeptides, anti-infective agents, present invention provides kits comprising the articles buffering agents, vitamins and minerals, analgesics, antico described above in a sterile package. agulants, coagulation factors, anti-inflammatory agents, 0040. In some embodiments of the present invention, the vasoconstrictors, vasodilators, diuretics, and anti-cancer compositions and methods described above enhance wound agents. In some embodiments, the wound active agent is healing. The present invention contemplates that wound heal dispersed in the polymer multilayer. In some embodiments, ing may be enhanced in a variety of ways. In some embodi the wound active agent is covalently associated with the poly ments, the compositions and methods minimize contracture mer multilayer. In some embodiments, the particles are func of the wound as to best favor function and cosmesis. In some tionalized. In some embodiments, the wound active agent is embodiments, compositions and methods promote wound attached to the particles. In some embodiments, the wound contracture to best favor function and cosmesis. In some active agent is contained within the particle. embodiments, the compositions and methods promote vascu 0044. In some embodiments, the polymer layer is dis larization. In some embodiments, the compositions and meth posed on a Support. In some embodiments, the Support is a ods inhibit vascularization. In some embodiments, the com polymeric Support. In some embodiments, the polymeric Sup positions and methods promote fibrosis. In some port is a polymer composition that is distinct from the poly embodiments, the compositions and methods inhibit fibrosis. mer multilayer. In some embodiments, the polymeric Support In some embodiments, the compositions and methods pro is an elastomeric polymer. In some embodiments, the poly mote epithelial coverage. In some embodiments, the compo meric Support and the polymer multilayer are separated by a sitions and methods inhibit epithelial coverage. In some water Soluble material. In some embodiments, the polymeric embodiments, the compositions and methods of the present Support is water Soluble. In some embodiments, the water invention modulates one or properties of cells in the wound soluble Support comprises polyvinyl alcohol. In some environment or in the immediate vicinity of the wound. The embodiments, the Support is a wound dressing. In some properties that are modulated, e.g., are increased or embodiments, the Support is a biologic wound dressing. In decreased, include, but are not limited to adhesion, migration, Some embodiments, the polymer multilayer is deposited onto proliferation, differentiation, extracellular matrix secretion, the Support by a method selected from the group consisting of phagocytosis, MMP activity, contraction, and combinations adsorption from solution and spin coating. thereof. 0045. In some embodiments, the particles have an elastic 0041. In some embodiments, the present invention pro modulus of from about 0.5 to about 10 GPa. In some embodi vides for the use of any of the compositions described above ments, the particles are interspersed or dispersed in the poly or elsewhere herein to enhance healing of a wound or modu mer multilayer. In some embodiments, the particles are dis late one or more properties of cells in the wound environment played on a Surface of the polymer multilayer. In some or in the immediate vicinity of the wound. In some embodi embodiments, the particles are underneath the polymer mul ments, the present invention provides for the use of a combi tilayer. In some embodiments, the particles are between the nation of a wound modifying agent and wound active agent to polymer multilayer and the Support. In some embodiments, enhance healing of a wound to enhance healing of a wound or the particles are on the surface of the polymer multilayer and modulate one or more properties of cells in the wound envi have a diameter that is greater than the thickness of the poly ronment or in the immediate vicinity of the wound. In some mer multilayer, and wherein the article further comprises an embodiments, the present invention provides for the use of antimicrobial silver composition. the articles described above to enhance healing of a wound or 0046. In some embodiments, the present invention pro modulate one or more properties of cells in the wound envi vides methods of modifying a surface comprising contacting ronment or in the immediate vicinity of the wound. a surface with a polymer multilayer comprising at least one 0042. In some embodiments, the present invention pro polymer, the polymer multilayer having associated therewith vides an article or composition comprising a polymer multi particles selected from the group consisting of nanoscale and layer comprising at least one polymer, the polymer multilayer microScale particles under conditions such that the polymer having associated therewith particles selected from the group multilayer is transferred to the surface. In some embodi consisting of nanoscale and microscale particles. In some ments, pressure is applied to the polymer multilayer to effect embodiments, the at least one polymer is selected from the transfer to the Surface. In some embodiments, the pressure is group consisting of a cationic polymer, an anionic polymer, a from about 10 to about 500 kPa. In some embodiments, the nonionic polymer an amphoteric polymer and combinations polymer multilayer is disposed on a Support. In some embodi thereof. In some embodiments, the polymer multilayer is ments, the support is from about 1 micrometer to about 10 from 1 nm to 500 nm thick, preferably from 1 nm to 250 nm mm in thickness. In some embodiments, the Surface is a thick. In some embodiments, the polymer multilayer has a wound dressing. In some embodiments, the wound dressing compliance of from 3 kPa to 500 kPa, 3 kPa to 10 MPa, or 3 is a biologic wound dressing. In some embodiments, the kPa to 5 GPa. In some embodiments, the particles are poly polymer multilayer is deposited on the Support by a process meric particles. In some embodiments, the particles are selected from the group consisting of adsorption from solu selected from the group consisting of spherical, ovoid, and tion, spraying, and spin coating. In some embodiments, the cylindrical shaped particles. In some embodiments, the diam Support is an elastomeric Support. In some embodiments, the eter of the particles is greater than the thickness of the poly Surface is a Soft or compliant Surface. In some embodiments, mer multilayer. the soft surface has an elastic modulus of from about 10 to 0043. In some embodiments, the articles or compositions about 100 kPa. In some embodiments, the soft surface is a comprise at least one wound active agent in association with biological Surface. In some embodiments, the biological Sur US 2011/O 1892.87 A1 Aug. 4, 2011
face is selected from the group consisting of skin, a wound microbial silver composition is releasable in an amount of bed, hair, a tissue surface, and an organ Surface. In some about 0.05 and 5ug/cm of the polymer multilayer per day. In embodiments, the biological Surface comprises molecules some embodiments, the releasable antimicrobial silver com selected from the group consisting of a proteins, a lipids, a position is releasable in an amount of about 0.05 and 2 ug/cm carbohydrates, and combinations thereof. In some embodi of the polymer multilayer per day. In some embodiments, the ments, the Surface comprises (e.g., is coated with or displays) releasable antimicrobial silver composition is releasable in an with glycosaminoglycans, collagen and/or hyaluronic acid. amount of about 0.05 and 1 g/cm of the polymer multilayer In some embodiments, the Surface is a Surface on a biomedi per day. In some embodiments, the releasable antimicrobial cal device. In some embodiments, the Surface is a silicone silver composition is releasable in an amount of about 0.1 to Surface. In some embodiments, the at least one polymer is 0.5 g/cm of the polymer multilayer per day. In some selected from the group consisting of a cationic polymer, an embodiments, the antimicrobial silver composition com anionic polymer, a nonionic polymeran amphoteric polymer prises silver nanoparticles. In some embodiments, the silver and combinations thereof. In some embodiments, the poly nanoparticles are Zerovalent silver nanoparticles. In some mer multilayer is from 1 nm to 500 nm thick. In some embodi embodiments, the composition provides at least 99.99% kill ments, the polymer multilayer has a compliance of from 3 kPa ing of Staphylococcus epidermis. In some embodiments, the to 500 kPa, 3 kPa to 10 MPa, or 3 kPa to 5 GPa. In some article does not substantially impair the healing of wounds. In embodiments, the particles are polymeric particles. In some Some embodiments, the antimicrobial silver composition per embodiments, the diameter of the particles is greater than the mits at least 90% viability of NIH-3T3 cells. In some embodi thickness of the polymer multilayer. In some embodiments, ments, the viability is assessed after 24h of incubation of the the particles are selected from the group consisting of spheri NIH-3T3 cells on the composition in the presence of growth cal, ovoid, and cylindrical shaped particles. In some embodi media. In some embodiments, the article is a film. In some ments, the polymer multilayer comprises at least one wound embodiments, the article is a three-dimensional object, at active agent. In some embodiments, the wound active agentis least a portion of which is coated with the polymer multilayer. selected from the group consisting of trophic factors, extra In some embodiments, the three-dimensional object is a cellular matrices, enzymes, enzyme inhibitors, defensins, medical device. polypeptides, anti-infective agents, buffering agents, Vita 0049. In some embodiments, the article is selected from mins and minerals, analgesics, anticoagulants, coagulation the group consisting of a polymeric Support, an elastomeric factors, anti-inflammatory agents, vasoconstrictors, vasodila Support, a wound care device, a wound dressing and a bio tors, diuretics, antimicrobial agents, growth factors, oli logic wound dressing. In some embodiments, the medical gopeptides, saccharides, polysaccharides, synthetic mol device partially contacts a subject. In some embodiments, the ecules comprising a biological moiety, metal particles, medical device is completely implanted in a Subject. In some electrodes, magnetic particles, honey, and anti-cancer agents. embodiments, the at least one of the polymers comprises In some embodiments, the wound active agent is dispersed in poly(allylamine hydrochloride). In some embodiments, at the polymer multilayer. In some embodiments, the wound least one of the polymers comprises poly(acrylic acid). In active agent is covalently associated with the polymer multi Some embodiments, the articles or compositions further com layer. In some embodiments, the particles are functionalized. prise at least one additional antimicrobial agent. In some embodiments, the wound active agent is attached to 0050. In some embodiments, the present invention pro the particles. In some embodiments, the particles have an vides a method for treating a wound comprising providing a elastic modulus of from about 0.5 to about 10 GPa. In some Subject with a wound and contacting the wound with an embodiments, the particles are dispersed in the polymer mul article or composition as described above. tilayer. In some embodiments, the particles are displayed on a 0051. In some embodiments, the present invention pro surface of the polymer multilayer. vides kits for the treatment of a wound in a Subject, compris 0047. In some embodiments, the transfer is done in the ing an article or composition as described above and instruc Substantial absence of a solution or a liquid solvent. In some tions for applying the article to the wound. In some embodiments, the transfer is a substantially or completely dry embodiments, the kits further comprise additional wound transfer. In some embodiments, the transfer is performed dressing material. In some embodiments, the wound dressing through a gas phase. In some embodiments, the transfer is material is a biologic wound dressing. performed in an environment where the humidity is less than 0052. In some embodiments, the present invention pro 100% of saturation. In some embodiments, the transfer is vides an article or composition comprising a solid Support performed in the absence of liquid water. comprising a releasable antimicrobial silver composition, 0048. In some embodiments, the present invention pro wherein the releasable antimicrobial silver composition is vides an article or composition comprising a polymer multi loaded atanamount of from 1 to about 500ug/cm of the solid layer loaded with an antimicrobial silver composition in the support and/or releasable in an amount of about 0.01 and 100 amount of about 100 to about 500 ug/cm of the polymer ug/cm of the solid support per day. In some embodiments, multilayer and/or having a releasable antimicrobial silver the solid Support is selected from the group consisting of a composition incorporated therein, wherein the releasable polymeric Support, an elastomeric Support, nanoparticles, antimicrobial silver composition is releasable in an amount of microparticles, a wound dressing and a biologic wound dress about 0.01 and 100 g/cm of the polymer multilayer per day. ing. In some embodiments, the releasable antimicrobial silver In some embodiments, the releasable antimicrobial silver composition is releasable in an amount of about 0.05 and 100 composition is releasable in an amount of about 0.05 and 100 ug/cm of the Solid support per day. In some embodiments, ug/cm of the polymer multilayer per day. In some embodi the releasable antimicrobial silver composition is releasable ments, the releasable antimicrobial silver composition is in an amount of about 0.05 and 20 g/cm of the solid support releasable in an amount of about 0.05 and 20 ug/cm of the per day. In some embodiments, the releasable antimicrobial polymer multilayer per day. In some embodiments, the anti silver composition is releasable in an amount of about 0.05 US 2011/O 1892.87 A1 Aug. 4, 2011
and 5ug/cm of the solid support per day. In some embodi In some embodiments, the antimicrobial silver composition ments, the releasable antimicrobial silver composition is comprises silver nanoparticles. In some embodiments, the releasable in an amount of about 0.05 and 2 g/cm of the silver nanoparticles are Zerovalent silver nanoparticles. In Solid Support per day. In some embodiments, the releasable Some embodiments, the antimicrobial silver composition antimicrobial silver composition is releasable in an amount of comprises multivalent silver ions carried by Zirconium phos about 0.05 and 1 g/cm of the solid support per day. In some phate. In some embodiments, the solid Support comprises a embodiments, the releasable antimicrobial silver composi material selected from the group consisting of a foam, a tion is releasable in an amount of about 0.1 to 0.5 g/cm of transparent thin film, a hydrogel, hydrofibers, hydrocolloids, the Solid Support per day. In some embodiments, the antimi alginate fibers, and combinations thereof. In some embodi crobial silver composition comprises silver nanoparticles. In ments, the Solid Support comprises a material selected from Some embodiments, the silver nanoparticles are Zerovalent the group consisting of Super absorbent polymers (SAP), silver nanoparticles. In some embodiments, the article does silica gel, Sodium polyacrylate, potassium polyacrylamides not substantially impair the healing of wounds. In some and combinations thereof. In some embodiments, the Solid embodiments, the articles or compositions further comprise Support comprises an absorbent material. In some embodi at least one additional antimicrobial agent. ments, the polymer multilayer is transferred to the Support in 0053. In some embodiments, the present invention pro the Substantial absence of a solvent. In some embodiments, vides methods for treating a wound comprising providing a the absorbent material is selected from the group consisting Subject with a wound and contacting the wound with the of a fabric, e.g., cotton or nylon gauze, and polymer foam. In article or composition as described above. Some embodiments, the absorbent material comprises a first 0054. In some embodiments, the present invention pro Surface comprising an anti-adherent material. In some vides methods for synthesizing a composition for treatment embodiments, the polymer multilayer is deposited on the first of a wound, comprising: a) assembling alternating layers of Surface comprising an anti-adherent material. In some polymers to form a film or coating and b) during the assem embodiments, the absorbent material comprises a second Sur bling, incorporating an antimicrobial silver composition into face fixed to an adhesive material. In some embodiments, the the alternating layers of polymers. adhesive material is sized to extend from one or more edges of 0055. In some embodiments, the present invention pro the absorbent material so that the absorbent material can be vides methods for synthesizing a composition for treatment exposed to the skin of a Subject. In some embodiments, the of a wound, comprising: a) assembling alternating layers of article further comprises a removable sheet, wherein the polymers to form a film or coating; b) incubating the film or article is removably affixed to the removable sheet. In some coating in bulk Solution of a silver salt; c) exposing the film or embodiments, the article is packaged in a sterile package. In coating to a reducing agent, and d) selecting films or coatings Some embodiments, the polymer multilayer has a thickness of for use having a silver loading level between 0.01 and 100 from about 1 nm to 500 nm. In some embodiments, the ug/cm. In some embodiments, at least one of the polymers polymer multilayer has a compliance of from about 3 to 500 comprises a solution containing poly(allylamine hydrochlo kPa. In some embodiments, the polymer multilayer com ride). In some embodiments, at least one of the polymers prises nanoscale to microscale particles incorporated therein. comprises a solution containing poly(acrylic acid). In some In some embodiments, the at least one wound active agent is embodiments, the bulk solution of a silver salt comprises an antimicrobial silver composition. In some embodiments, silver nitrate. In some embodiments, the concentration of Ag" the silverloading of the polyelectrolyte multilayer is between in the silver nitrate solution is from 0.005 mM to 5 mM. In about 0.01 and 500 ug/cm of the antimicrobial silver com Some embodiments, the pH of the poly(acrylic acid) solution position. In some embodiments, the antimicrobial silvercom is from 2.5 to 7.5. position comprises silver nanoparticles. In some embodi 0056. In some embodiments, the present invention pro ments, the silver nanoparticles are selected from the group vides a Solid Support; and a polymer multilayer Supported by consisting of Zerovalent silver nanoparticles and multivalent the Solid Support, the polymer multilayer comprising at least silver ions (carried by Zirconium phosphate). one wound active agent. In some embodiments, the article 0057. In some embodiments, the solid support comprises a further comprises a water soluble material between the solid biologic wound dressing. In some embodiments, the biologic Support and the polymer multilayer. In some embodiments, wound dressing comprises (e.g., is coated with or displayS) a the Solid Support comprises a polymer. In some embodiments, biological molecule selected from the group consisting of the polymer is an elastomeric polymer. In some embodi collagen, hyaluronic acid, glycosaminoglycans, keratin, ments, the elastomeric polymer is a silicon polymer. In some fibronectin, vitronectin, laminin, and combinations or frag embodiments, the silicon polymer is selected from the group ments thereof. In some embodiments, the biopolymer is Sup consisting of polydimethylsiloxane and medical grade sili ported on a silicone film. In some embodiments, the silicone cone. In some embodiments, the polymer multilayer has a film comprises a fabric Support. In some embodiments, the thickness of from about 1 nm to 500 nm. In some embodi fabric Support is a nylon fabric Support. In some embodi ments, the polymer multilayer has a compliance of from ments, the polymer multilayer is deposited on the silicone about 3 kPa to 5 GPa, preferably about 3 kPa to 500 kPa. In film. In some embodiments, the polymer multilayer has a Some embodiments, the polymer multilayer comprises thickness of from about 1 nm to 500 nm. In some embodi nanoscale to microscale particles incorporated therein. In ments, the polymer multilayer has a compliance of from Some embodiments, the particles have a diameter that is about 3 to 500 kPa. In some embodiments, the polymer mul greater than thickness of the polymer multilayer. In some tilayer comprises nanoscale to microscale particles incorpo embodiments, the at least one wound active agent is an anti rated therein. In some embodiments, the particles have a microbial silver composition. In some embodiments, the sil diameter that is greater than the thickness of the polymer verloading of the polyelectrolyte multilayer is between about multilayer. In some embodiments, the at least one wound 0.01 and 500 g/cm of the antimicrobial silver composition. active agent is an antimicrobial silver composition. In some US 2011/O 1892.87 A1 Aug. 4, 2011
embodiments, the silver loading of the polyelectrolyte mul (0071. The term “wound” refers broadly to injuries to the tilayer is between about 0.01 and 500 ug/cm of the antimi skin and Subcutaneous tissue initiated in different ways (e.g., crobial silver composition. In some embodiments, the anti pressure sores from extendedbed rest and wounds induced by microbial silver composition comprises silver nanoparticles. trauma) and with varying characteristics. The methods and In some embodiments, the silver nanoparticles are selected compositions described herein are useful for treatment of all from the group consisting of Zerovalent and multivalent silver types of wounds, including wounds to internal and external nanoparticles. tissues. Wounds may be classified into one of four grades depending on the depth of the wound: i) Grade I: wounds DESCRIPTION OF THE FIGURES limited to the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade III: wounds extending into the subcu 0058 FIG. 1 provides a schematic of a wound bed modi taneous tissue; and iv) Grade IV (or full-thickness wounds): fied with a polyelectrolyte multilayer. wounds wherein bones are exposed (e.g., a bony pressure 0059 FIG. 2 provides a schematic of a wound bed modi point Such as the greater trochanter or the sacrum). fied with covalent modifying agents. (0072. The term “partial thickness wound” refers to 0060 FIG. 3 exemplifies the covalent immobilization of wounds that encompass Grades I-III; examples of partial proteins to model amine-terminated treated glass Surfaces. thickness wounds include burn wounds, pressure Sores, 0061 FIG. 4 shows ex vivo results for multilayer deposi venous stasis ulcers, and diabetic ulcers. The term “deep tion of polyelectrolytes polystyrene sulfonate (PSS) and wound' is meant to include both Grade III and Grade IV FITC-labelled poly (allylamine hydrochloride) (FITC-PAH). wounds. The present invention contemplates treating all 0062 FIG.5 demonstrates the difference in healing of full wound types, including deep wounds and chronic wounds. thickness cutaneous wounds in diabetic (db/db) mice com 0073. The term “chronic wound refers to a wound that pared to control (wild type) mice. has not healed within 30 days. 0063 FIG. 6 is exemplary of the use of beads for healing a 0074 The phrases “promote wound healing, "enhance wound bed. In this example, carboxylic acid-terminated wound healing,” and the like refer to either the induction of beads are activated outside the wound bed by using NHS and the formation of granulation tissue of wound contraction EDC. The activated beads are introduced into the wound bed and/or the induction of epithelialization (i.e., the generation and are immobilized in the wound bed via reaction of the of new cells in the epithelium). Wound healing is conve activated Surfaces of the beads with amine groups present in niently measured by decreasing wound area. the wound bed. Growth factors are introduced into the wound 0075. The term “wound active agent” refers to known or bed and immobilized via reaction with the exposed surfaces potential chemical compounds that induce a desired pharma of the activated beads. cological, physiological effect useful in the treatment and 0064 FIG.7 provides a list of antimicrobial polypeptides, healing of a wound, wherein the effect may be prophylactic or identified by name and AMSDb database ID number. therapeutic. The terms also encompass pharmaceutically 0065 FIG.8 shows the viability of NIH-3T3 mouse fibro acceptable, pharmacologically active derivatives of those blasts after 24hr incubation in growth media on silver-loaded active agents specifically mentioned herein, including, but polyelectrolyte multilayers (PEMs) (Example 13). not limited to, trophic factors, extracellular matrices, 0066 FIG. 9 shows viable bacteria detected on silver enzymes, enzyme inhibitors, defensins, polypeptides, anti loaded PEMs 8 hr after incubation with 10 cfu/ml of S. infective agents (including but not limited to ionic silver, epidermidis in buffer. PEMs with varying silver loading were elemental silver, and silver nanoparticles), buffering agents, prepared by incubating them with serial decimal dilutions of Vitamins and minerals, analgesics, anticoagulants, coagula bulk Ag" solution. PEMs incubated with 5 mM Ag" (silver tion factors, anti-inflammatory agents, vasoconstrictors, nitrate) solution as indicated in this figure contained -0.38 vasodilators, diuretics, and anti-cancer agents. ug/cm (where no cytotoxicity towards NIH-3T3 cells was 0076. The term “polymer multilayer” refers to the compo observed in FIG. 8), while silver loading in rest of the PEMs sition formed by sequential and repeated application of poly was below the detection limit of the elemental analysis spec mer(s) to form a multilayered structure. For example, poly trometer. electrolyte multilayers are polymer multilayers are formed by 0067 FIG. 10 shows NIH 3T3 cells after 24h incubation the alternating addition of anionic and cationic polyelectro on glass surfaces coated with PEMs (10.5 bilayers of PAH, lytes to a wound or support. The term “polymer multilayer” 5/PAA) without silver (a,c,e) or with silver nanoparticles at also refers to the composition formed by sequential and concentrations (b) 0.62, (d) 0.48, or (f) 0.39 ug/cm. PEMs repeated application of polymer(s) to a wound or to a solid were prepared with PAA solution of pH 5.5 (a,b), or 6.5 (c,d) support. In addition, the term “polymer layer can refer to a or 7.5 (e, f). single layer composed of polymer molecules, such as anionic 0068 FIG. 11 provides a schematic depiction of one or cationic polyelectrolyte molecules, existing either as one embodiment of polymer multilayers with microscale beads. layer within multiple layers, or as a single layer of only one 0069 FIG. 12 provides a graph depicting transfer of a type of polyelectrolyte molecules on a wound or Support. polymer multilayer containing microscale beads to a soft While the delivery of the polymers to the wound bed or Surface as compared to transfer of a polymer multilayer with Support is sequential in preferred embodiments, the use of the out microscale beads. term “polymer multilayer is not limiting in terms of the resulting structure of the coating. It is well understood by DEFINITIONS those skilled in the art that inter-diffusion of polymers such as polyelectrolytes can take place leading to structures that may 0070. To facilitate an understanding of the invention set be well-mixed in terms of the distribution of anionic and forth in the disclosure that follows, a number of terms are cationic polyelectrolytes. It is also understood that the term defined below. polyelectrolyte includes polymer species as well as nanopar US 2011/01892.87 A1 Aug. 4, 2011 ticulate species, and that it is not limiting in scope other than carbon unit. Primary amines have the general formula RNH to indicate that the species possesses multiple charged or and examples include, but are not limited to, aniline, methy partially charged groups. It is also well understood by those lamine, and 1-propylamine. skilled in the art that multilayer structures can be formed I0086) The term “DNA delivery agent” refers to any mol through a variety of interactions, including electrostatic inter ecule that can bring DNA into contact with an identified actions and others such as hydrogen bonding. Thus, the use of target. In some instances, a DNA delivery agent causes uptake the term “polyelectrolyte” is not limiting in terms of the of DNA into a cell or cells, in vitro or in vivo. DNA delivery interactions leading to the formation of the wound bed con agents can be viruses including, but not limited to, adenovi StructS. ruses and retroviruses. DNA delivery agents can also be non 0077. The term “crosslinked herein refers to a composi viral agents including, but not limited to, plasmids, lipids, tion containing intermolecular crosslinks and optionally liposomes, polymers and peptides. intramolecular crosslinks as well, arising from the formation 0087. The term “exposable” refers to anything that is of covalent bonds. Covalent bonding between two crosslink capable of being exposed. An exposable surface or molecule able components may be direct, in which case an atom in one is one that is made available to interaction with other surfaces component is directly bound to an atom in the other compo or molecules. For example, in the context of the present nent, or it may be indirect, through a linking group. A invention, a covalent modification agent is exposable to a crosslinked structure may, in addition to covalent bonds, also wound active agent; thus, the two agents can interact with include intermolecular and/or intramolecular noncovalent each other and form covalent bonds. bonds such as hydrogenbonds and electrostatic (ionic) bonds. 0088. The term “functionalized’ refers to a modification 0078. The term “covalent modification agent” refers to of an existing molecular segment to generate or introduce a any molecule that covalently links molecules to each other. new reactive functional group (e.g., a maleimido or succin Covalent modification agents include homobifunctional and imidyl group) that is capable of undergoing reaction with heterobifunctional cross-linkers as well as photoactivatable another functional group (e.g., a sulfhydryl group) to form a cross linkers. covalent bond. For example, a component containing car 0079. The term “homobifunctional cross-linker” refers to boxylic acid (-COOH) groups can be functionalized by a molecule used to covalently link identical or similar mol reaction with N-hydroxy-succinimide or N-hydroxysulfos ecules to each other. Homobifunctional cross-linkers have uccinimide using known procedures, to form a new reactive two identical reactive groups; thus, a homobifunctional cross functional group in the form of an activated carboxylate linker can only link molecules of the same type to each other. (which is a reactive electrophilic group), i.e., an N-hydrox Conversely, a "heterobifunctional cross-linker” refers to a y succinimide ester or an N-hydroxysulfosuccinimide ester, molecule used to covalently link dissimilar molecules to each respectively. In another example, carboxylic acid groups can other, because it has two or more different reactive groups that be functionalized by reaction with an acyl halide, e.g., an acyl can interact with various molecules of different types. Het chloride, again using known procedures, to provide a new ero- and homo-multifunctional crosslinkers refers to multi reactive functional group in the form of an anhydride. valent crosslinkers with both hetero- and homo-crosslinking 0089. As used herein, the term “aqueous solution functionalities. Activated dendrimers are an example of mul includes solutions, suspensions, dispersions, colloids, and the tifunctional crosslinkers. like containing water. 0080. The term “subject” refers to any animal (e.g., a 0090. As used herein, the term "click chemistry” refers to mammal), including, but not limited to, humans, non-human the use of chemical building blocks with built-in high-energy primates, rodents, dogs, cats, and the like, which is to be the content to drive a spontaneous and irreversible linkage reac recipient of a particular treatment. Typically, the terms 'sub tion with appropriate complementary sites in other blocks. ject' and “patient” are used interchangeably herein. These chemical reactions (e.g., including, but not limited to. 0081. The term “surfactant” refers to an amphiphilic mate those between azide and acetylene groups that combine rial that modifies the surface and interface properties of liq readily with each other) are specific and result in covalent uids or solids. Surfactants can reduce the surface tension linkage between the two molecules. between two liquids. Detergents, wetting agents, emulsifying 0091. The term “native chemical ligation” refers to a agents, dispersion agents, and foam inhibitors are all surfac chemoselective reaction of two unprotected peptide seg tantS. ments. The reaction results in an initial thioester-linked spe 0082. The term “block copolymer refers to a polymer cies, then spontaneous rearrangement of this transient inter consisting of at least two monomers. In a block copolymer, mediate occurs, yielding a full-length product with a native adjacent blocks are constitutionally different, i.e. adjacent peptide bond at the ligation site. blocks comprise constitutional units derived from different 0092. The term “specific protein binding” refers to an species of monomer or from the same species of monomer but interaction between two or more proteins that have high affin with a different composition or sequence distribution of con ity and specificity for each other. Proteins must bind to spe stitutional units. A block copolymer can be thought of as two cific other proteins in vivo in order to function. The proteins homopolymers joined together at the ends. are required to bind to only one or a few other proteins of the 0083. The term “solvent refers to a liquid that can dis few thousand proteins typically present in Vivo: these inter solve a substance. The term “organic solvent” refers to a actions are employed in vitro in the present invention to attach solvent derived from a petroleum-based product. wound active agents to the wound. In the context of the I0084. The term “polyelectrolyte” refers to a water-soluble present invention, specific protein binding interactions macromolecular polymer substance containing many repeat include, but are not limited to, those between biotin and ing ionic constituent units, including cations and anions. avidin, neutravidin, or streptavidin; glutathione-S-transferase 0085. The term “primary amine” refers to a derivative of and glutathione; and nickel-nitrilotriacetic acid and polyhis ammonia in which a hydrogen has been replaced by a hydro tidine. US 2011/O 1892.87 A1 Aug. 4, 2011
0093. The term “device” refers to an object that contacts BIOBRANETM, IntegraTM, Apligraf R, Dermagraft(R), the body or bodily fluid of a subject for therapeutic or pro Oasis.R., Transcyte R, Cryoskin R and Myskin R. phylactic purposes. Some devices may partially or indirectly 0099. As used herein, the term “antimicrobial silver com contact the body or bodily fluid of a subject (e.g., catheter, position” refers to a composition that comprises silver as an dialysis tubing, diagnostic sensors, drug delivery devices), active antimicrobial agent. Examples of “antimicrobial silver while other devices are completely imbedded in or encom compositions' include, but are not limited to silver nanopar passed by the body of a subject (e.g., stent, pacemaker, inter ticles, elemental silver, Zero Valent silver, multivalent silver nally implanted defibrillator, angioplasty balloon, orthopedic ions carried by Zirconium phosphate (ZP-Ag)(See, e.g., device, spinal cage, implantable drug pump, artificial disc, ear Wound Repair and Regeneration, 16: 800-804), and silver containing compounds such as silver Sulfadiazine and related disc). compounds. The term “releasable antimicrobial silver com 0094. The term “selective toxicity” refers to the property position” refers to a antimicrobial silver composition that can of differentially toxic effects on mammalian versus microbial be released from a material, for example, a polymer multi cells. For example, a selectively toxic agent may effectively layer Solid Support, so that antimicrobial activity can be kill bacterial cells while permitting growth and viability of observed. The release of the antimicrobial silver composition mammalian cells. can be defined as an amount of the composition released from 0095. The term “toxic' refers to any detrimental or harm a defined area or volume of the material. ful effects on a Subject, a cell, or a tissue as compared to the same cell or tissue prior to the administration of the toxicant. DETAILED DESCRIPTION OF THE INVENTION 0096. As used herein, the terms "nanoparticle' and 0100. The complex nature of wounds, and the lack of "nanoscale particles' are used interchangeably and refer to a significant clinical progress based on current therapies, indi nanoscale particle with a size that is measured in nanometers, cates the urgent need for new and unconventional approaches. for example, a nanoscopic particle that has at least one dimen The microenvironment of the pathologic/chronic wound bed sion of less than about 1000, 500, or 100 nm. Examples of is dysregulated with alterations in extracellular matrix con nanoparticles include nanobeads, nanofibers, nanohorns, stituents, degradative enzymes, growth factor and other cyto nano-onions, nanorods, and nanoropes. active factor activity. The present invention provides compo 0097. As used herein, the term “microparticle' and sitions and methods for engineering of the wound bed itself “microscale particles are used interchangeably and refers to by, for example, altering the Surface chemistry/structure of a microscale particle with a size that is measured in microme the woundbed to promote favorable cell behaviors that accel ters, for example, a microscale particle that has at least one erate wound healing. In some embodiments, the woundbed is dimension of less than about 10 micrometers, 5 micrometers, first treated with a priming agent (i.e., primer) that provides a or 2 micrometers. uniform, reactive bed on the wound surface. The primed 0098. The term “wound dressing refers to materials wound bed is then treated with a desired agent, such a wound placed proximal to a wound that have absorbent, adhesive, active agent. protective, osmoregulatory, pH-regulatory, or pressure-in 0101. In normal wound healing, the coordinated interplay ducing properties. Wound dressings may be in direct or indi between fibroblasts, vascular cells, extracellular matrix com rect contact with a wound. Wound dressings are not limited by ponents and epithelial cells results in a seamless progression size or shape. Indeed, many wound dressing materials may be through an inflammatory reaction, wound repair, contracture cut or configured to conform to the dimensions of a wound. and coverage by an epithelial barrier. However, in many Sub Examples of wound dressing materials include but are not jects with dysregulated wound microenvironment, systemic limited to gauze, adhesive tape, bandages, and commercially disease or other confounding circumstances, the wound heal available wound dressings including but not limited to adhe ing processes become asynchronous resulting in an indolent sive bandages and pads from the Band-Aid R. line of wound ulcer (Pierce, 2001, Am. J. Pathol. 159:399). In other sub dressings, adhesive bandages and pads from the NeXcare(R) jects, a loss or lack of regulatory responses to appropriately line of wound dressings, adhesive bandages and non-adhesive modulate cellular behaviors during healing causes an exuber pads from the Kendall Curity Tefla R. line of wound dressings, ant proliferative response that in itself is a problem for the adhesive bandages and pads from the Tegaderm R) line of subject. This is particularly true for patients prone to keloid wound dressings, adhesive bandages and pads from the Steri formation or in burn patients where excessive fibroblastic Strip(R) line of wound dressings, the COMFEEL(R) line of proliferation and collagen production result indisfiguring and wound dressings, adhesive bandages and pads, the Duo disabling scar formation. dermR line of wound dressings, adhesive bandages and pads, 0102. It is clear that across the spectrum of non-healing the TEGADERMTM line of wound dressings, adhesive ban wounds that there are a variety of inciting mechanisms and dages and pads, the OPSITE(R) line of wound dressings, adhe wound microenvironments. These wounds exhibit pathology sive bandages and pads, and biologic wound dressings. A at many junctures in the progression to closure. Deficits in “biologic wound dressing is a type of wound dressing that angiogenesis, fibroblastic responses and re-epithelialization comprises, e.g., is coated with or incorporates, cells and/or all play a role in chronic wounds. Thus, a single factor treat one or more biomolecules or fragments of biomolecules that ment approach to chronic wounds is not likely to be fruitful can be placed in contact with the wound surface. The biomol across the disparate array of wounds presented in the clinical ecules may be provided in the form of an artificial tissue milieu. In Such a heterogeneous environment, a more prom matrix. Examples of Such biomolecules include, but are not ising strategy involves the identification of compounds that limited, to collagen, hyaluronic acid, glycosaminoglycans, are able to modulate specific aspects of the cellular response laminin, vitronectin, fibronectin, keratin, antimicrobial and behavior in the wound environment, providing the poten polypeptides and combinations thereof. Examples of suitable tial for custom crafting of a healing response tailored to the biologic wound dressings include, but are not limited to, individual wound and patient. US 2011/O 1892.87 A1 Aug. 4, 2011
0103 Due to the heterogeneous spectrum of wound envi ness, and porosity, it is also possible to incorporate in the film ronments, it is contemplated that stimulating a “desirable' architecture functionalized macromolecules (Caruso et al., healing response is best achieved by a differential modulation 1997, Langmuir 13:3427; Cassier et al., 1998, Supramol. Sci. of the cellular responses within the wound. The present inven 5:309). It has also been demonstrated that the layer-by-layer tion provides for the selection of a subset of cytoactive com deposition process is not limited in applicability to polyelec pounds from a list of candidates for immobilization into the trolytes, but can be applied to viruses, nanoparticles, non wound bed so as to differentially modulate the endothelial, ionic polymers, proteins and other forms of microscopic and fibroblastic and epithelial components within the healing nanoscopic matter. A recent review provides information on a response. As such, the present invention provides the poten wide range of species and interfacial structures that can be tial to achieve high quality healing responses in a variety of formed by the layer-by-layer deposition procedure. The clinical conditions, thereby providing a strategy for engineer scope of the invention described herein is not limited to poly ing the wound bed for personalized therapeutics for each electrolytes but applies to all species that have been demon unique wound healing challenge encountered by a clinician. strated to be incorporated into interfacial structures by the Specific examples where differential modulation of the cel layer-by-layer deposition process. lular responses within the wound would yield substantial 0107 The present invention provides a variety of embodi benefit include chronic wounds in diabetics or venous stasis ments for altering the composition of the wound bed. In some ulcers, where pancellular promotion of healing responses is embodiments, a wound modifying agent is applied to prime desired but in particular a vibrant angiogenic response is the wound bed. As described in detail below, wound modify needed to Support all of the other aspects of wound healing. In ing agents are agents that applied to a wound bed and either other wounds, where the strength of the healed wound is a key covalently or noncovalently modify the wound bed. concern, modulation to promote a fibroblastic response with Examples of wound modifying agents include homobifunc a normal angiogenic and epithelial component is desirable. tional and heterobifunctional linkers, polyelectrolytes, non 0104. In contrast, in some burns, such as deep second ionic polymers, combinations of polyelectroloytes and non degree burns where dermal and hair shaft epithelial elements ionic polymers, and nano- and micro-particles including persist to replace lost tissues, a richangiogenic and epithelial beads and needles. In some embodiments, the wound modi response is needed, but it is desirable to mitigate the fibro fying agent alters a property of the wound bed selected from blastic reaction to reduce scar hypertrophy, contracture and the group consisting of compliance, pH, alkalinity, and oxi disfigurement. A similar mitigation is desirable in healing dative or reductive strength, net charge, hydrophilicity, subjects prone to keloid formation where the proliferative osmotic strength, nanoscale or Submicron topographic fea fibroblastic response in these wounds must be Suppressed. It tures, electroconductivity, MMP production, phagocytosis, is also advantageous in wounds near joints or orifices to be and transglutaminase activity. In preferred embodiments, the able to promote rapid healing and coverage with epithelium wound modifying agents are used to incorporate wound but modulate the fibroblastic response so that improved active agents so that the wound active agents are localized to suppleness of the tissue is retained. Modulation of the fibro the wound bed. The wound active agents can be covalently or blastic response in this way has the potential to provide Supe noncovalently attached to the wound modifying agent. Fur rior clinical outcomes and reduce the need for Subsequent thermore, the wound active agents may form a gradient via reconstructive procedures targeted at recovery of limb or the wound modifying agent. In further embodiments, the other critical bodily functions. The feasibility of such an wound modifying agents alter the compliance of the wound approach has been demonstrated previously, such as in the bed. In these embodiments, the polymers or nano- or micro report by Muehlberger and colleagues on the effects tretinoin particles have a predetermined hardness. In further embodi on incisional wounds (Muehlberger et al., 2005, J. Am. Acad. ments, the compliance gradients with varying levels of hard Derm. 52:583). In that study, application of tretinoin resulted ness may be formed by the polymers, nano- or micro in an increased fibroblastic proliferation but the production of particles. collagen was diminished. 0108. In some embodiments of the present invention, the 0105. The modification of surfaces has become an impor wound active agent is silver or a form of silver. Silver is a tant challenge in the last decade for applications in implant widely used nonspecific biocidal agent that acts against a very materials, prostheses, and artificial organs, allowing broad broad spectrum of bacterial species (Yin et al., 1999, J. Burn medical applications for implant and tissue engineering Care Rehabil. 20:195; herein incorporated by reference in its (Langer and Vacanti, 1993, Science 260:920); Peppas and entirety), yeast species, fungal species (Wright et al., 1999, Langer, 1994, Science 263:1715; Angelova and Hunkeler, Am. J Infect. Control 27:344; herein incorporated by refer 1999, Trends Biotechnol. 17:409). For the improved integra ence in its entirety), and viruses (Hussain et al., 1991, Bio tion efficiency of implants, several approaches, involving the chem. Biophys. Res. Comm. 189: 1444; herein incorporated alteration of physicochemical, morphological, and biochemi by reference in its entirety), including several antibiotic resis cal properties of device Surfaces, have been investigated in an tant strains (Wright et al., 1998, Am. J. Infect. Control 26:572; effort to obtain a suitable bone implant interface. Self-as herein incorporated by reference in its entirety). However, sembled monolayers (SAMs) or Langmuir Blodgett tech topical silver agents undergo fast inactivation in the wounds niques have been commonly employed to produce new inter because of the highly reactive nature of silver ions. Frequent faces (Mrksich, 1997, Curr. Opin. ColloidInterface Sci. 2:83: wound dressings result in large excess of silver being deliv Losche, 1997, Curr. Opin. Solid State Mater. Sci. 2:546). ered to the wound, resulting intoxic effects from overdose. In 0106 More recently, a new versatile method of self vitro studies have shown cytotoxic effects of silver ions on assembled architectures based on the alternate deposition of fibroblasts (Poon et al., 2004, Burns 30:140; Hildago et al., polyanions and polycations has been developed for the Skin Pharmacol. Appl. Skin Physiol. 11:140; each herein buildup of multilayered polyelectrolyte films (Decher, 1997, incorporated by reference in its entirety), keratinocytes (Poon Science 277: 1232). Besides varying film thickness, rough et al., 2004, Burns 30:140; herein incorporated by reference US 2011/O 1892.87 A1 Aug. 4, 2011 in its entirety), hepatocytes (Baldi et al., 1988, Toxicol. Lett. 0111. The silver-nanoparticles impregnated thin films of 41:261; herein incorporated by reference in its entirety), and some embodiments of the present invention exploit different lymphocytes (Hussain et al., 1991, Biochem. Biophys. Res. principles for delivering silver into the wound-matrix. The Comm. 189: 1444; herein incorporated by reference in its films can be applied directly on the exposed wound-bed, entirety). allowing them to release the silver ions locally into the wound. This reduces the amount of bactericidal silver needed 0109 The highly reactive nature of silver ions complicates to impregnate into the film by several orders compared to their delivery to wound bed. The released silver ions become topical silver delivery agents, significantly reducing the sil rapidly inactive as they readily bind to proteins and chloride ver-based toxicity. While the present invention is not limited within complex wound fluid (Mooney et al., 2006, Plastic and to any particular mechanism, and an understanding of the Reconstr. Surg. 1 17:666; herein incorporated by reference in mechanism is not necessary to practice the present invention, its entirety), resulting in unwanted adsorption of silver ions in it is contemplated that while silver-impregnated dressings epidermis cells and sweat glands (Klasen, 2000, Burns and ointments have to deliver high levels of silver into the 26:117; herein incorporated by reference in its entirety). Sil wound bed to overcome rapid consumption of silver ions in ver based topical solutions like 0.5% silver nitrate, or oint wound-fluid that limits the penetration of released silver, the ments like 1% silver sulfadiazine cream, that are currently localized release of silver right into the wound-matrix by used in clinics for wound healing, release silver at concentra Some polymeric thin film embodiments presented herein cir tions up to 3200 ppm but most of this is rapidly inactivated cumvents the use of large loadings of silver. though the formation of chemical complexes in the wound 0112 Some molecularly-thin composite film embodi (Dunn et al., Burns, 2004, 30(supplement 1): S1; herein incor ments of the present invention can be tuned to contain as low porated by reference in its entirety), necessitating frequent as 0.4 g/cm of soluble silver and release a total of less than applications, up to 12 times a day for silver nitrate solutions 1 ppm silver ions in buffers. Such films display 99.9999% and at least twice a day with silver sulfadiazine creams. Fre bacteria-killing efficiency without any measurable cytotoxic quent dressings result in large excess of silver being delivered ity to mammalian cells. Furthermore, films containing these to the wound, resulting intoxic effects and discoloration from levels of silver allow adherence and growth of mammalian overdose (Atiyehet al., 2007, Burns 33:139; Trop et al., 2006, cells (e.g., NIH-3T3 mouse fibroblasts). In some embodi J. Trauma 60:648; each hereinincorporated by reference in its ments, the releasable antimicrobial silver composition is entirety). Wound-dressings with silver incorporated into the loaded at an amount of from 1 or 10 to about 50, 100, 200, dressing itself have been recently introduced. They provide 300, 400, 500 or 1000 g/cm of the polymer multilayer prolonged release of silver to the wound, allowing dressings and/or releasable in an amount of about 0.01 and 100 ug/cm to be changed less frequently. However, these reservoir-dress of the polymer multilayer per day. In some embodiments, the ings have to be impregnated with large amount of silver, releasable antimicrobial silver composition is releasable in an which results in cytotoxicity. Silver released from a leading amount of about 0.05 and 100 ug/cm of the polymer multi commercial wound-dressing ActicoatTM, that contains nanoc layer. In some embodiments, the releasable antimicrobial rystalline silver (Dunn et al., Burns, 2004, 30(supplement silver composition is releasable in an amount of about 0.05 1):51; herein incorporated by reference in its entirety), was and 20 g/cm of the polymer multilayer. In some embodi shown to be toxic to in vitro monolayer cell cultures of kera ments, the releasable antimicrobial silver composition is tinocytes and fibroblasts (Poon et al., 2004, Burns 30:140; releasable in an amount of about 0.05 and 5 g/cm of the Trop et al., 2006, J. Trauma 60:648; each herein incorporated polymer multilayer. In some embodiments, the releasable by reference in its entirety). Thus, the challenges with current antimicrobial silver composition is releasable in an amount of topical silver antimicrobials lie in their low silver release about 0.05 and 2 g/cm of the polymer multilayer. In some levels, the lack of penetration, the rapid consumption of silver embodiments, the releasable antimicrobial silver composi ions, and the presence of silver nitrate or cream bases that are tion is releasable in an amount of about 0.05 and 1 g/cm of pro-inflammatory, negatively affecting wound healing. Issues the polymer multilayer. In some embodiments, the releasable like wound staining, electrolyte imbalance, and patient dis antimicrobial silver composition is releasable in an amount of comfort also exist. about 0.1 to 0.5 g/cm of the polymer multilayer. 0110. In certain embodiments, the present invention com 0113 Experiments conducted during the course of devel prises novel methods for providing controlled and localized oping some embodiments of the present invention allowed loadings of non-cytotoxic levels of antimicrobial silver com systematic variation of the concentration of silver in the films positions to Surfaces that circumvents using excess-loading of and demonstrated their cytotoxicity and anti-bacterial effi silver in the wound while retaining anti-bacterial activity and ciency to be dependent on the silver-loading. In comparison, reduced cytotoxicity. In experiments conducted during the the amount of soluble silver in the commercial wound dress course of developing certain embodiments of the present ing ActicoatTM is ~100 ug/cm (Dunn et al., 2004, Burns invention (Example 13), nanometer-thick polymeric films 30(supplement 1):S1; herein incorporated by reference in its were assembled that were impregnated with silver nanopar entirety). ActicoatTM releases a total of over 120 ppm silver on ticles to levels resulting in efficient killing of bacteria but no dissolution in water for 24 hr (Taylor et al., 2005, Biomate cytotoxic effects on the adherence and growth of mouse fibro rials 26:7221; herein incorporated by reference in its blasts cells on the films. In some embodiments, the polymeric entirety), and has been shown to be cytotoxic to keratinocytes films are impregnated with an antimicrobial silver composi and fibroblasts (Poon et al., 2005, Burns 30:140; Trop et al., tion by incubating the film in an antimicrobial silver compo 2006, J. Trauma 60:648; each herein incorporated by refer sition as described in Example 13. In other embodiments, the ence in its entirety). antimicrobial silver composition is directly incorporated into 0114 Several reports exist on composite films of polyelec the polymer multilayer during synthesis of the multilayer. trolytes that incorporate silver and display anti-bacterial See, e.g., Langmuir. 2005 Dec. 6; 21 (25): 11915-21. activity by releasing silver in the solution (Lee et al., 2005, US 2011/O 1892.87 A1 Aug. 4, 2011
Langmuir 21:9651; Liet al., 2006, Langmuir 22:9820; Grun using, for example, polyelectrolyte multilayers or beads. FIG. lanet al., 2005, Biomacromolecules 6:1149; Yu et al., 2007, 1 provides a schematic diagram 100 of a wound bed 110 on Bioconj. Chem. 18:1521; Shi et al., 2006, J. Biomed. Mat. which a polyelectrolyte multilayer 130 has been deposited. Res. Part A 76A:826; each herein incorporated by reference The diagram 100 depicts that the wound bed 110 comprises a in its entirety). However, all these studies have focused on heterogeneous surface depicted by shapes 120 which repre controlling the Volume fraction and concentration of silver in sent different chemical moieties. The polyelectrolyte multi the films. Only films containing high loadings of silver (-5.5 layer 130 provides a homogenous surface onto which func ug/cm or more) have been shown to demonstrate anti-bacte tional groups can 140 can be attached to form a homogenous rial activity (Wang et al., 2002, Langmuir 18:3370; Logaret functionalized surface 150. In the embodiment depicted, the al., 2007, Nanotechnol. 18:325.601; each herein incorporated functional groups are uniform, however, in Some preferred by reference in its entirety). Such polyelectrolyte films, when embodiments, different functional groups are utilized. A wide loaded with those levels of silver, display strong cytotoxicity variety of active agents can then be attached to the Surface via and kill 100% NIH-3T3 cells seeded on them (e.g., Example the functional groups 140. In other embodiments, the wound 13). bed is covalently modified with covalent modification agents. 0115. In experiments conducted during the course of The covalent modification agents include, but are not limited developing certain embodiments of the present invention to, homobifunctional and heterobifunctional cross-linkers as (e.g., Example 13), loading levels of silver in PEMS were well as photoactivatable cross linkers. FIG.2 provides a sche identified and achieved that lead to silver-impregnated anti matic diagram 200 of a wound bed 210 comprising a hetero bacterial molecularly-thin films permitting adhesion of mam geneous surface depicted by shapes 220 which represent dif malian cells without exhibiting cytotoxicity. The films find ferent chemical moieties. The wound bed is covalently use for the localized delivery of bactericidal silver to the modified by reacting covalent modification agents 230 with wounds, reducing risks of toxicity from silver overdose the different chemical moieties to provide a relatively homog observed with other topical delivery agents. In some embodi enous functionalized surface 250. In preferred embodiments, ments, implantable medical devices can be coated with these covalent modification agents 230 present functional groups silver-impregnated thin films to kill bacteria right during their 240. In the embodiment depicted, the functional groups are initial attachment to these Surfaces and prevent bacterial colo uniform, however, in some preferred embodiments, different nization, a leading cause of implant failure. functional groups are utilized. A wide variety of active agents 0116. Thus, the silver-releasing moleculary-thin compos can then be attached to the Surface via the functional groups ite films of some embodiments of the present invention have 240. These embodiments are discussed in more detail below. advantage over: 1) other topical delivery agents of silver 0119. It is contemplated that the wound bed is an currently used in clinics; 2) wound-dressings that contain extremely heterogeneous environment. The compositions large amounts of silver in them; and 3) other bactericidal and methods of the present invention are designed to modify composite thin-films containing large amounts of silver, the wound bed to provide a homogenous environment that which are cytotoxic to mammalian cells and do not allow provides for uniform and predictable delivery or uniform mammalian cells to adhere and grow over them. incorporation of active agents into the wound bed. Surpris 0117 Using silver-nanoparticle molecularly-thin films for ingly, it has been found that modification of wound beds in localized delivery of bactericidal silver in wounds can over this manner greatly reduces the amount of active agent which come silver-related toxicity encountered with topical silver is needed; i.e., the effective amount of active agent needed is delivery agents for wound healing (Poon et al., 2004, Burns reduced. Surface functionalization allows for precise control 30:140; Baldiet al., 1988, Toxicol. Lett. 41:261; Atiyeh et al., of the amount of active agent used or delivered and further 2007, Burns 33:139; Coombs et al., 1992, Burns 18:179; each allows for the formation of gradients of active agents on the herein incorporated by reference in its entirety). Furthermore, wound bed. In some preferred embodiments, the priming these polyelectrolyte multilayers (PEMs) are uniform, highly agent is optimized to react with the wound bed. In some interpenetrated ultrathin nanocomposite films, typically far embodiments, the priming agent provides an optimized Sur less than 1 um thick (Decher, 1997, Science 277: 1232; Ham face for enhanced or optimized delivery of an active agent. In mond, 1999, Curr. Opin. Colloid Interface Sci. 4:430; each Some embodiments, a chemical parameter of the wound bed herein incorporated by reference in its entirety). Their porous is changed. For example, the wound bed may be modified to and Supramolecular architecture allows incorporating a vari be more or less acidic, basic, alkaline, reducing, or oxidizing ety of molecules including DNA, enzymes, viruses, dendrim or have a higher or lower ionic strength. ers, colloids, inorganic particles, and dyes (Decher, 1997, 0.120. There is a wide array of candidate molecules for Science 277: 1232; herein incorporated by reference in its improving healing chronic wounds. For example, basement entirety). Thus, along with delivering bactericidal silver, in membrane constituents and growth factors promote wound Some embodiments they can be used for simultaneous local healing. In some embodiments, the extracellular matrices ized delivery of other bioactive agents in the wound-matrix deposited and immobilized are constituents of native base (e.g., cell recruiting growth factors, DNA plasmids encoding ment membrane. Native ECMs comprise a mixture of glyco such growth factors). Since PEMs can conformally coat sub proteins, proteoglycans and hyaluronic acid. These glycopro strates of any type, size or shape, including natural and Syn teins and proteoglycans include, but are not limited to, fibrin, thetic polymers (Hammond, 1999, Curr. Opin. ColloidInter elastin, fibronectin, laminins, glycosaminoglycans, nidogens face Sci. 4:430; herein incorporated by reference in its and collagens. Cytoactive factors such as growth factors are entirety), they can be constructed on any wound-bed or also part of ECMs. In some embodiments, extracellular implantable medical device. matrix components, such as collagen, laminin, glycosami 0118. In one embodiment, the present invention provides noglycans, or hyaluronic acid are deposited and immobilized for the deposition and immobilization of cytoactive factors on a wound bed. In some embodiments, a synthetic matrix and extracellular matrices (ECMs) on the wound bed by such as MATRIGELTM is deposited on a woundbed. MATRI US 2011/O 1892.87 A1 Aug. 4, 2011
GELTM is a commercially available basement membrane like charge (i.e., non-ionic polymers) or a combination of charged complex that retains many characteristics of a native base and uncharged polymer layers. In some embodiments, it is ment membrane, including a three-dimensional nanoscale contemplated that polyelectrolyte films built-up by the alter topographic Surface. The present invention is not limited to a nated adsorption of cationic and anionic polyelectrolyte lay particular mechanism. Indeed, an understanding of the ers constitute a novel and promising technique to modify mechanism is not necessary to practice the present invention. wound surfaces in a controlled way (Decher et al., 1992, Nonetheless, it is contemplated that the nanoscale topo Thin Solid Films 210/211:831; Decher, 1997, Science 277: graphic features of a basement membrane modulate funda 1232). One of the most important properties of such multi mental cell behaviors including migration, adhesion, prolif layers is that they exhibit an excess of alternatively positive eration and differentiation (Abrams et al., 2002, Biomimetic Materials and Design: Interactive Biointerfacial, Tissue Engi and negative charges (Caruso et al., 1999, JAm Chem Soc neering, and Drug Delivery, Eds. Dillow and Lowman; Diehl 121:6039; Ladam et al., 2000, Langmuir 16:1249). Not only et al., 2005, J. Biomed. Mater. Res. A 75:603; Foley et al., can this constitute the motor of their buildup (Joanny, 1999, 2005, Biomaterials 26:3639: Karuri et al., 2004, J. Cell Sci. Eur. Phys.J. Biol. 9:117), but it allows, by simple contact, to 117:3153; Liliensiek et al., 2006, J. Biomed. Mater. Res. A adsorb a great variety of compounds such as dyes, particles 79:185). In some embodiments, the present invention further (Cassagneau et al., 1998, J. Am. Chem. Soc. 120:7848; provides methods, formulations, compositions and kits for Caruso et al., 1999, Langmuir 15:8276; Lvov et al., 1997, altering the compliance of the wound Surface. In some Langmuir 13:6195), clay microplates (Ariga et al., 1999, embodiments, the local compliance (the compliance that cells Appl. Clay Sci. 15:137) and proteins (Keller et al., 1994, J. see) is altered by immobilizing a thin layer of extracellular Am. Chem. Soc. 116:8817; Lvov et al., 1995, J. Am. Chem. matrix constituents such as MATRIGELTM or an appropriate Soc. 117:61 17: Caruso et al., 1997, Langmuir 13:3427). hydrogel or other synthetic matrix or by enzymatic treatment 0.125. In some embodiments, the polymer multilayers, of the wound bed. In other embodiments, compliance is Such as polyelectrolyte multilayers, are nanoscale in dimen altered by the addition of cross-linking agents to the wound Sion. Accordingly, in Some embodiments, the polymer mul bed to cross-link components already presenting the wound bed, or components deliberately introduced into the wound tilayers are from about 1 nm to 1000 nm thick, from about 1 bed. It has also been demonstrated by Picart and coworkers nm to 500 nm thick, from about 1 nm to 100 nm thick, from that it is possible to control the compliance of multilayer about 1 nm to about 25 nm thick, from about 1 nm to about 10 structures formed from polyelectrolytes by the addition of nm thick, or less than about 500 nm, 100 nm, 25 nm or 10 nm. cross-linking agents. thick. It is contemplated that the nanoscale dimension of the 0121. In some embodiments, the cytoactive factors that polymer multilayers (i.e., the nanoscale thickness) allows for are deposited and immobilized on a wound bed include, but the loading of a lower total amount of an active agent while are not limited to, those factors that are mitogenic for epithe still allowing delivery of an effective amount (i.e., an amount lial cells and vascular endothelial cells and other factors that of active agent that accelerates wound healing as compared to are elements in wound closure. For example, in Some embodi controls) of the active agent as compared to matrix structures ments, growth factors such as platelet derived growth factor with greater thickness. It is contemplated that the lower total (PDGF), and/or epidermal growth factor (EGF), known to be loading levels result in reduced toxicity in the wound envi mitogenic for epithelial cells are deposited in a wound bed. In ronment, especially when antimicrobial compounds are other embodiments, vascular endothelial growth factor incorporated into the polymer multilayer. (VEGF), known to be mitogenic for vascular endothelial I0126. In some embodiments, the compliance of the poly cells, comprise the cytoactive factors immobilized on the mer multilayers is adjusted to facilitate cell migration in the wound bed. It is contemplated that the present invention is not wound. In some embodiments, the polymer multilayers limited by the ECM components or cytoactive factors immo exhibit a compliance, measured in kilopascals (kPa) of from bilized on the wound bed, indeed any extracellular matrix about 3 to about 500 kPa, about 7 to about 250 kPa, about 10 components and/or cytoactive factor that improves wound to about 250 kPA or from about 10 to about 200 kPa. healing is equally applicable. Additional cytoactive and O127 1. Formation of Polyelectrolyte Layers wound active agents that can be incorporated are provided I0128. The cationic polyelectrolyte poly(L-lysine) (PLL) below. interacts with anionic sites on cell Surfaces and in the extra cellular matrix (Elbert and Hubbell, 1998, J. Biomed. Mater. I. Priming the Wound Bed Res. 42:55). In some embodiments, the present invention 0122. In some embodiments, the present invention pro provides a method of treating a wound with the sequential vides compositions and methods for priming the woundbed application of a cationic polyelectrolyte, an anionic polyelec to provide uniform reactive surface for later modification. trolyte, and a wound active agent to the wound. In other Suitable compositions for priming a wound bed include, but embodiments, the treatment includes the sequential and are not limited to, polyelectrolytes and chemical crosslinking repeated application of a cationic polyelectrolyte, an anionic agents that react covalently with functional groups found in polyelectrolyte, and a wound active agent to the wound. wound beds Such as carboxy, thiol, amide and Sugar groups. I0129 Polyelectrolyte layers are formed by alternating (0123. A. Multilayer Deposition on Wound Bed applications of anionic polyelectrolytes and cationic poly 0.124. In some embodiments, the present invention pro electrolytes to surfaces to form a polyelectrolyte layer. The vides compositions, formulations, methods and kits for layers can be used to deliver a wound active agent to a wound. depositing a multilayer structure on a wound bed. In some Preferably, at least four layers, and, more preferably, at least embodiments, the multilayer structures comprise layers of six layers are used to form the polyelectrolyte multilayer. polymers that form polyelectrolytes, while in other embodi 0.130. The method of treatment of the present invention is ments, the multilayers comprise polymers that do not have a not limited to use on a wound surface. The formation of a US 2011/O 1892.87 A1 Aug. 4, 2011 polyelectrolyte layer that includes a wound active agent may (histidine), and nonpeptide polyamines such as poly(ami beformed on any surface to which delivery of a wound active nostyrene), poly(aminoacrylate), poly (N-methylaminoacry agent is desirable. late), poly (N-ethylaminoacrylate), poly(N,N-dimethyl 0131. In some embodiments, the cationic polyelectrolyte aminoacrylate), poly(N,N-diethylaminoacrylate), poly(ami used is PLL and the anionic polyelectrolyte used is poly(L- nomethacrylate), poly(N-methyl amino-methacrylate), poly glutamic acid) (PGA). Indeed, the use of a variety of poly (N-ethyl aminomethacrylate), poly(N,N-dimethyl ami electrolytes is contemplated, including, but not limited to, nomethacrylate), poly(N,N-diethyl aminomethacrylate), poly(ethylene imine) (PEI), poly(allylamine hydrochloride) poly(ethyleneimine), polymers of quaternary amines, such as (PAH), poly(sodium 4-styrenesulfonate) (PSS), poly(acrylic poly(N.N.N-trimethylaminoacrylate chloride), poly(methy acid) (PAC), poly(maleic acid-co-propylene) (PMA-P), poly acrylamidopropyltrimethylammonium chloride), and natural (acrylic acid) (PAA), and poly(vinyl sulfate) (PVS). It is also or synthetic polysaccharides such as chitosan. In some possible to use naturally occurring polyelectrolytes, includ embodiments, PLL is a preferred material. ing hyaluronic acid and chondroitin Sulfate. 0.138. In general, the polymers must include at least five 0.132. In still further embodiments, the polymer is a den charges, and the molecular weight of the polycationic mate drimer, grafted polymer, or star architecture polymer. In other rial must be sufficient to yield the desired degree of binding to embodiments, the multilayer responds to or is organized in a tissue or other Surface, having a molecular weight of at least the presence of an electric field, for example an electric field 1000 g/mole. formed by placing electrodes on either side of a wound. 3. Anionic Polymers 0.133 Referring to FIG.1, in some embodiments, the poly 0.139 mer multilayer 130 can be comprised of polystyrene sul 0140 Polyanionic materials useful in the present inven fonate, an amphiphillic polyelectrolyte with an affinity for tion can be any biocompatible water-soluble polyanionic hydrophobic regions of the wound bed, and an amphoteric polymer, for example, any polymer having carboxylic acid polymer. The polymer multilayer is preferably functionalized groups attached as pendant groups. Suitable materials include with one or more crosslinking agents presenting an alkyne so alginate, carrageenan, furcellaran, pectin, Xanthan, hyalu that a uniform surface is presented (e.g., 140 in FIG. 1 where ronic acid, heparin, heparan Sulfate, chondroitin Sulfate, der X represents an alkyne group). From this point, widely avail matan Sulfate, dextran Sulfate, poly(meth)acrylic acid, oxi able click chemistries can be used to add desired wound active dized cellulose, carboxymethyl cellulose and agents to the modified Surface of the wound. In some embodi crosmarmelose, synthetic polymers and copolymers contain ments, the wound modifying agent is an azide conjugate or ing pendant carboxyl groups, such as those containing maleic otherwise comprises an azide group and is reacted with the acid or fumaric acid in the backbone. Polyaminoacids of alkyne groups displayed on the wound bed in a Huisgen predominantly negative charge are also Suitable. Examples of Cycloaddition. these materials include polyaspartic acid, polyglutamic acid, 0134. Other suitable methods for preparing polyelectro and copolymers thereof with other natural and unnatural lyte multilayers include those described, for example, in Cho amino acids. Polyphenolic materials such as tannins and and Char, Langmuir 20:4011-4016, 2004: Okamura et al., lignins can be used if they are sufficiently biocompatible. Adv. Mater. 21,4388-92 (2009), Cho et al., Adv. Mat. 13(14): Preferred materials include alginate, pectin, carboxymethyl 1076-1078 (2001); and U.S. pat. Publ. 2010/0062258; the cellulose, heparin and hyaluronic acid. entire contents of each of which is incorporated herein by 0141 4. Nonionic Polymers reference. Suitable methods include layer by layer deposi 0142. In some embodiments, the multilayer structures are tion, formation on SAMs, and spin coating assisted assembly. formed from uncharged polymers or from a combination of 0135 2. Cationic Polymers charged and uncharged polymers. Examples of uncharged 0.136 Cationic polymers useful in the present invention polymers include, but are not limited to, dextran, dextran can be any biocompatible water-soluble polycationic poly sulfate, diethylaminoethyl (DEAE)-dextran, hydroxyethyl mer, for example, any polymer having protonated hetero cellulose, ethyl(hydroxyethyl) cellulose, acrylamide, poly cycles attached as pendant groups. As used herein, “water ethylene oxide, polypropylene oxide, polyethylene oxide— soluble” means that the entire polymer must be soluble in polypropylene oxide copolymers, PAAN, Ficoll, polyvi aqueous solutions, such as buffered saline or buffered saline nylpyrolidine, and polyacrylic acid. with Small amounts of added organic solvents as co-solvents, 0143 5. Amphoteric Polymers at a temperature between 20 and 37° Centigrade. In some 0144. In some embodiments, the multilayer structures are embodiments, the material will not be sufficiently soluble formed from one or more amphoteric polymers, alone in (defined hereinas soluble to the extent of at least one gram per combination with the other polymers described herein. In liter) in aqueous solutions per se but can be brought into Some embodiments, the amphoteric polymers comprise one Solution by grafting the polycationic polymer with water or more of acrylic acid (AA), DMAEMA (dimethylaminoet soluble polynonionic materials such as polyethylene glycol. hyl methacrylate). APA (2-aminopropyl acrylate), Mor 0.137 Representative cationic polymers include natural phEMA (morpholinoethyl methacrylate), DEAEMA (diethy and unnatural polyamino acids having net positive charge at laminoethyl methacrylate), t-ButylAEMA neutral pH, positively charged polysaccharides, and posi (t-butylaminoethyl methacrylate), PipEMA (piperidinoethyl tively charged synthetic polymers. Examples of Suitable methacrylate), AEMA (aminoethyl methacrylate), HEMA polycationic materials include polyamines having amine (2-hydroxyethyl methacrylate), MA (methyl acrylate), MAA groups on either the polymer backbone or the polymer side (methacrylic acid) APMA (2-aminopropyl methacrylate), chains, such as poly-L-lysine (PLL) and other positively AEA (aminoethyl acrylate). In some embodiments, the charged polyamino acids of natural or synthetic amino acids amphoteric polymer comprises (a) carboxylic acid, (b) pri or mixtures of amino acids, including, but not limited to, mary amine, and (c) secondary and/or tertiary amine. The poly(D-lysine), poly(ornithine), poly(arginine), and poly amphoteric polymers have an isoelectric point of 4 to 8, US 2011/O 1892.87 A1 Aug. 4, 2011 preferably 5 to 7 and have a number average molecular weight uefied petroleum gases like propane, isobutane, and N-butane in the range of 10,000 to 150,000. and mixtures thereof Dimethyl ether is another propellant. 0145 6. Application of Multilayers Compressed gas propellants that are preferably non-toxic, 0146 The wound modifying agent, such as a polymer non-flammable, and inert can be used. Examples include multilayer, can be applied to the wound by a variety of meth carbon dioxide, nitrous oxide and N and the like. Mixtures of ods. In some embodiments, it is contemplated that the poly the above are often used. mer or polymer multilayers, is applied, preferably sequen 0151. The quantity of propellant used is critical only in tially, to the wound using either a pump (including Syringes, that if an insufficient amount is used, the driving force to expel inkjet printers, and electrojets) or aerosol spray. In other the entire composition from the container will be lacking embodiments, particle bombardment is utilized. In other Generally, the composition will comprise from 75% to 95% embodiments, the use of a brush including an air brush is by weight propellant. contemplated. In other embodiments, a sponge is utilized. In 0152. In some embodiments, the invention contemplates a other embodiments a Solid Support or stamp Such as an elas kit comprising a container with a cationic polyelectrolyte, a tomeric material, for example, PDMS (polydimethylsilox second container containing an anionic polyelectrolyte, and a ane), silicone, hydrogel or latex, is used to Support the wound third container containing at least one wound active agent. In modifying agents and mechanically transfer the agent into the still other embodiments, in addition to a container with a wound bed. In these embodiments, the polymer multilayers cationic polyelectrolyte, a second container containing an are pre-formed on the stamp. In further embodiments, nano anionic polyelectrolyte, and a third container containing at or micro-particles are arranged on the stamp for delivery to least one wound active agent, the kit provides an application the wound. In other approaches, electric fields or magnetic device for administering the polyelectrolytes and at least one fields are used to facilitate transfer of the wound modifying wound active agent to the wound. In some preferred embodi agents into the wound bed. ments, the containers comprise a propellant. In other embodi 0147 In some embodiments, the polymers are applied to a ments, the containers comprise a pump. wound by a spray, such as via a pump or aerosol device. In 0153. In some embodiments, contacting comprises deliv Some embodiments, the polymer layers are applied sequen ery of the polymer and the at least one wound active agent to tially. In one embodiment, a solution, dispersion, or Suspen the wound using a reaction chamber dressing (RCD). In some sion of the wound modifying agent and wound active agent is embodiments, it is contemplated that the RCD will comprise sprayed onto the wound to form the polyelectrolyte layer. In a solid barrier adhered to an area completely Surrounding the embodiments where the wound modifying agent and wound wound. Within the solid barrier reside inflow and outflow active agent are Supplied as aerosols, a propellant is used to orifices to which tubes from solution reservoirs can be con provide the force for expulsion from the container. In some nected. The use of a variety of solid barriers is contemplated, embodiments, the wound modifying agent or the wound including, but not limited to, Gortex, latex, Teflon, PVDF, active agent and the propellant form a single liquid phase so plastic and rubber. that the composition is delivered consistently. 0154. In some embodiments, the wound modification 0148. In general, for aerosol delivery, the ingredients of agents are applied to the wound by microfluidics printing, the composition are mixed to form a Substantially homog microstamping (U.S. Pat. Nos. 5.512,131 and 5,731,152, enous solution, slurry, dispersion, or the like. For two-part both of which are incorporated by reference herein in their systems, each part is mixed. The compositions are placed in entirety), or microcontact printing (PCT Publication WO an appropriate container and the propellant is added using 96/29629, incorporated by reference herein in its entirety). In conventional techniques, such as cold filling or pressure fill other embodiments, the wound modifying agents are applied ing techniques. The composition can be delivered using a to the wound via a pulsejet Such as an inkjet printer. Examples commercially available aerosol sprayer Such as, for example, of suitable inkjet printers for application of biological fluids the PrevalTM aerosol spray unit available from Precision Valve include those described in U.S. Pat. No. 7,128,398, WO Corporation, NY, USA, which has a modular power unit and 95/25116 and WO 98/41531, each of which is incorporated refillable containerjar. The propellant is a mixture of propane, by reference in its entirety. In other embodiments, the wound isobutane, and dimethyl ether. modifying agents are applied by a drop dispensers such as the 014.9 The composition can also be delivered using a tip of a pin or in an open capillary and, touch the pin or Syringe outfitted with a spray head, or a dual spray device capillary to the surface of the substrate. Such a procedure is outfitted with a spray head and, optionally, a mixing chamber. described in U.S. Pat. No. 5,807,522. When the fluid touches The device may include a meter so that the quantity of applied the surface, some of the fluid is transferred. In other embodi composition can be controlled. ments, the wound modifying agents are applied be pipetting, 0150. Any of a number of propellants known to those micro-pipetting or positive displacement pumps such as the skilled in the art can be used, provided that it is chemically Biodot equipment (available from Bio-Dot Inc., Irvine Calif., inert to the other ingredients of the composition. Suitable USA). propellants include vinyl chloride and mixtures of vinyl chlo 0155 One or more wound active agents are preferably ride and dichlorodifluoromethane, other fluorochlorohydro applied to the wound along with the wound modification carbons known as the Freons and the Genetrons, and blends of agent. In some embodiments, the wound active agent is fluorochlorohydrocarbons, chlorinated hydrocarbons, and covalently attached to the wound modification agent. In other hydrocarbons. Examples of fluorochlorohydrocarbons embodiments, the wound active agent is incorporated into the include trichloromonofluoromethane, dichlorodifluo wound modifying agent, for example, by application during romethane, dichloromonofluoromethane, 2-tetrafluoroet sequential application of polymer layers in a polymer multi hane, 1,1-dichloro-1,2,2-tetrafluoroethane, 1-chloro-1,1-di layer. In other embodiments, the wound active agents are fluoroethane, 1,1-difluoroethane, and octofluorocyclobutane, delivered by a microarrayer, pipette, positive displacement and mixtures thereof Examples of hydrocarbons include liq pump, inkjet printer, microneedle array, elastomeric stamp, US 2011/O 1892.87 A1 Aug. 4, 2011 20 gene gun, electrojet, or airbrush as described above in rela and non-spherical particles contain their own Surface topog tion to the wound modifying agents. raphy. For example, the bead Surface may comprise undula B. Modification of Wound Beds with Biocompatible Particles tions, indentations, tunnels, holes, knobs, ridges, etc. Spheri 0156. In some embodiments, the wound modifying agent cal particles, such as beads, may or may not be inert and may, is a nano- or micro-particle. In some embodiments, nanom for example, be magnetic. Magnetic beads comprise, for eter to Submicrometer sized biocompatible particles. Such as example, a magnetic core or magnetic particles. Examples of spherical (e.g., beads) and/or non-spherical (e.g., oblongs, different bead compositions are found at, for example, U.S. needles, cubes, tetrahedral, mushroom-like structures, hay Pat. Nos. 5,268,178, 6,458,858, 6,869,858, and 5,834,121, bale-like structures) particles or electrospun polymers, are each of which is incorporated herein by reference in its applied (e.g., either directly or indirectly) to the wound bed, entirety. thereby creating a three-dimensional topographic woundbed. 0159. In some embodiments, biocompatible particles of For example, application of biocompatible particles to a the present invention used for wound healing are coated with wound bed results in the creation of knobs, ridges, spikes, layers of polyelectrolytes, ECM components, antimicrobials, undulations, and the like in the wound bed. Microbeads have proteins with hydrolysable bonds, proteins with enzymati a size generally ranging from about 1 to about 500 microme cally cleavable bonds, etc. and other wound healing compo ters, while nanobeads have a size generally ranging from sitions wherein the Surface compliance of the beads is main about 1 to about 1000 nanometers. Microbeads may further tained for optimal wound healing. For example, the comprise micro- (i.e., from 1 to 100 micrometers) or nano compliance of the layers for wound healing as deposited on scale (0.1 to 1000 nanometer) features on the surface of the the biocompatible particles are contemplated to be of optimal bead. Nanobeads may further comprise nanoscale features stiffness (e.g., not too soft, not too hard) Such that cells within (i.e., 0.1 to 500 nanometer features) on the surface of the bead. the wound bed are capable of optimal anchorage and spread In some embodiments, a wound active agent is present in the ing. Engler et al. (2004, Biophys. J. 86:617-628) discusses form of a bead or particle. Such as silver nanoparticles. Substrate compliance for cell anchorage and spreading. In 0157. In some embodiments, the biocompatible particles Some embodiments, the biocompatible particles are layered are biodegradable. In some embodiments, the biocompatible with polyelectrolytes, ECM components, antimicrobials, etc. particles are not biodegradable. The biocompatible particles, wherein substrate stiffness of at least 1 kPa, at least 5 kPa, at for example, are modified with Surface chemistry cross-link least 8 kPa, at least 10 kPa, at least 20 kPa, at least 40 kPa, at ers that allow attachment and immobilization of wound active least 60 kPa, at least 80 kPa is realized as measured using the agents, extracellular matrix compounds, polyelectrolytes, methods of Engler et al. (2004). In preferred embodiments, etc. as described further herein. It is contemplated that the Substrate compliance of the wound healing compositions as incorporation of modified biocompatible particles, for applied to biocompatible particles is at least 8 kPa to about 80 example, facilitates the presentation of ECM constituents that kPa or 100 kPa. It will be recognized that hardness within Support epithelial coverage of the wound, provides cytoactive these limit are contemplated without specifically listing all factors for wound healing, and introduces topographic fea such hardnesses, for example, 10, 20, 30, 40, 50, 60, or 70 kPa tures that Support cellular behaviors for promoting wound limits as upper or lower ranges are all contemplated by the healing. In some embodiments, the biocompatible particles present invention. In some embodiments, the ECM composi are functionalized with mesoscopic cross-linkers to, for tion preferentially used in conjunction with polyelectrolytes, example, broadly enable covalent chemistry in the wound antimicrobials, and other wound healing compositions is col bed. In some embodiments, cross-linkers are attached to lagen. negatively charged biocompatible particles. In some embodi 0160. In some embodiments, the layers as applied to a ments, the biocompatible particles comprise layers of mul wound bed or to a biocompatible particle comprise gradients tiple different constituents for wound healing, or a single of wound healing compositions, for example, wound active constituent for wound healing. For example, spherical par agents. For example, the concentrations of the compositions ticles Such as beads placed in a wound bed comprise, or upon are layered in a wound bed or on a biocompatible particle in which are layered, one or more intermixed species of poly a gradient Such that higher concentrations of a particular electrolytes, ECM agents, proteins with hydrolysable bonds, composition is greater proximal to the wound bed than distal proteins with enzymatically cleavable bonds, and the like for to the wound bed in a vertical fashion. The converse, where wound healing as previously described. In some embodi concentrations of compositions is greater distal to the wound ments, antimicrobials, either alone or in combination with bed than proximal, is also contemplated. Concentration of other wound healing compositions, are also applied to the compositions in a wound bed or on a biocompatible particle biocompatible particles. Antimicrobials include, but are not wherein a horizontal gradient is deposited is also contem limited to, antibiotics, metal based antimicrobial composi plated. Topographical gradients are also contemplated, tions comprising silver (e.g., ionic silver, elemental silver, wherein compositions are deposited Such that the concentra silver nanoparticles), copper, selenium, triclosan-based anti tions of compositions in a wound bed or on a biocompatible microbials, thiabendazole-based antimicrobials, isothiazoli particle follow the topography of the Substrate, for example, a none-based antimicrobials, zinc-pyrithione-based antimicro higher concentration of compositions is deposited in the val bials, and/or 10'-oxybisphenoxarsine-based antimicrobials leys of undulations of an exemplary Substrate compared to the (OBPA). The present invention is not limited by the type of peaks of the undulations. Likewise, the present invention antimicrobial used. contemplates that compliance gradients can be formed in the 0158. In some embodiments, the size of the biocompatible woundbed by controlled application of micro- or nano-beads particles, such as beads, are at least lnm, at least 5 nm, at least of varying hardness. For example, beads having one, two, 10 nm, at least 20 nm, at least 50 nm, at least 100 nm, at least three, four or more different hardnesses are applied to form a 200 nm, at least 300 nm, at least 500 nm, or at least 800 nm in Vertical gradient (i.e., a depth gradient) on the wound or a diameter. In some embodiments, beads and other spherical horizontal gradient (i.e., a gradient along the length or width US 2011/O 1892.87 A1 Aug. 4, 2011
of the wound), or a combination of vertical and horizontal ing compositions, etc. The present invention is not limited by gradients. Horizontal and vertical gradients can also be the constituents that make up a layer of deposition on a formed with beads functionalized with 1, 2, 3, 4 or more biocompatible particle, and many permutations of wound different wound active agents. See Ichinose et al., Biomate healing compositions and biocompatible particle layering rials. Volume 27, Issue 18, June 2006, Pages 3343-3350; would be apparent to a skilled artisan. Stefonek et al., Immobilized gradients of epidermal growth 0164. In some embodiments, the deposition of layers in factor promote accelerated and directed keratinocyte migra the wound bed or on a biocompatible particle or other sub tion. Wound Repair and Regeneration (OnlineEarly Articles). strate to be added to a wound bed is accomplished by “stamp doi:10.11111.1524-475X.2007.00288.x: Kapur et al., Immo ing, also referred to as "contact printing”. A stamp Substrate bilized concentration gradients of nerve growth factor guide is any Substrate that can be used to transfer one or more than neurite outgrowth. Journal of Biomedical Materials Research one entity or composition (e.g., polyelectrolytes, ECM com Part A. Volume 68A. Issue 2, Pages 235-243: DeLong et al., ponents, proteins with hydrolyzable or enzymatically cleav Covalently immobilized gradients of bFGF on hydrogel scaf able bonds, etc.) that is (are) covalently or non-covalently folds for directed cell migration. Biomaterials 26 (2005) bound to the surface of the stamp substrate to another surface. 3227-3234; Liu, et al., 2007 Sep 25; 17892312 Endothelial Examples of Suitable stamp Substrates include, but are not Cell Migration on Surface-Density Gradients of Fibronectin, limited to, polydimethylsiloxane (PDMS) and other elasto VEGF, or Both Proteins. meric (e.g., pliable) materials. In some embodiments, differ 0161 In some embodiments, wound healing compositions ent concentrations of the same wound healing composition applied to the wound bed or biocompatible particles (e.g., are arrayed in different areas of the stamp substrate. In other beads, needles, etc.) are time released. Such that wound heal embodiments, a variety of different compositions are arrayed ing compositions that are applied to the wound bed or bio on the stamp Substrate surface. In some embodiments, mul compatible particles are released over a period of time, for tiple wound healing compositions in multiple concentrations example 3 hours, 5 hours, 10 hours, 1 day, several days, one are arrayed on the stamp Substrate Surface. The wound heal week, several weeks, etc. thereby providing short and/or long ing compositions are then introduced to the stamp Substrate term treatment for the wound bed. Surface, which are in turn stamped into the woundbed or onto 0162. In some embodiments, biocompatible particles used another Substrate for introduction into the wound bed (e.g., in woundbed healing applications of the present invention are biocompatible particles). not, for example, spherical as previously described, but take 0.165. In other embodiments, the biocompatible particles, on shapes such as oblongs, needles, mushroom-like struc for example needles, are coated and/or layered with wound tures, haybale-like structures, etc. In some embodiments, the healing compositions, anti-microbials, proteins that are biocompatible particle shape is preferably needles. The hydrolyzably cleavable, proteins that are enzymatically manufacture of needles useful in embodiments of the present cleavable, etc., and applied to a pad which is Subsequently invention can be accomplished through microfabrication applied to a wound. Pads include, but are not limited to, techniques, such as those found in, for example, McAllisteret band-aids, gauze, Surgical packings, and the like. In some al., 2003, Proc. Natl. Acad. Sci. 100:13755-13760. In some embodiments, the pad is impregnated with wound healing embodiments, biocompatible particles are coated and/or lay compositions, anti-microbials, proteins that are hydrolyzably ered with wound healing compositions, anti-microbials, pro cleavable, proteins that are enzymatically cleavable, etc. In teins that are hydrolyzably cleavable, proteins that are enzy Some embodiments, the biocompatible particles (e.g., cov matically cleavable, antimicrobials, etc. as described herein. ered and/or layered with wound healing compositions, anti In some embodiments, the coated and/or layered biocompat microbials, proteins that are hydrolyzably cleavable, proteins ible particles are applied to a wound bed by stamping, spray that are enzymatically cleavable, etc.) are affixed in a detach ing, or other methods as described herein. able or non-detachable manner to the pad prior to applying the 0163. In some embodiments, the layers of wound healing pad to a wound. In some embodiments, when the pad is compositions as described herein are deposited on a wound removed from the wound the biocompatible particles are bed or on biocompatible particles by electrostatic bonding. In retained (e.g., not detached) on the pad, whereas in other other compositions, non-electrostatic interactions are uti embodiments when the pad is removed from the wound the lized, such as van der Waals interactions, metal ion-ligand nanostructures are retained (e.g., detached) in the wound bed. coordination or hydrogen bonding. In some embodiments, For example, a pad is impregnated with compositions as the layers of wound healing compositions as described herein described and further affixed with biocompatible particles are deposited by piezoelectric application, using methods and that are themselves coated and/or layered with compositions systems as described in, for example, U.S. Pat. No. 6,368,079 as described for wound healing. The pad with biocompatible and Sumerel et al., 2006, Biotechnol. J. 1:976-987. In other particles is applied to the wound and while the pad is Super embodiments, the wound healing compositions are directly ficial to the wound bed, the biocompatible particles thereon or indirectly deposited into the wound bed by using electro reach down in proximity with the wound bed as compared to jetting technologies, including electroextrusion, and electro the location of the pad. The pad releases (e.g., either over a spraying and electrospinning. In some embodiments, the lay short period of time or in a time-release manner) the impreg ers deposited comprise one or more wound healing nated wound healing compositions on or near the Surface of compositions. In some embodiments, the layers are biocom the wound whereas the affixed biocompatible particles patible particles (e.g. spherical beads, needles, and the like) release the coated and/or layered wound healing composi intermixed with wound healing compositions. In other tions (e.g., either over a short period of time or in a time embodiments, layered deposition is sequential. For example, release manner) more proximal to the wound bed. Alterna sequential deposition of wound healing compositions alone, tively, a pad with affixed biocompatible particles (both pad or sequential deposition of wound healing compositions fol and nanostructures comprising wound healing compositions lowed by biocompatible particles, followed by wound heal as previously described) is first applied to a wound bed. The US 2011/O 1892.87 A1 Aug. 4, 2011 22 pad is subsequently removed; however the detachable bio is described in U.S. Pat. No. 5,807,522. When the fluid compatible particles remain in the wound bed. The pad serves touches the surface, some of the fluid is transferred. In other not only to deliver wound healing compositions to a wound embodiments, the wound modifying agents are applied be bed, but further serves a purpose of delivering biocompatible pipetting, micro-pipetting or positive displacement pumps particles, such as needles, into a wound bed. The biocompat such as the Biodot equipment (available from Bio-Dot Inc., ible particles left in the wound bed, for example, continue Irvine Calif., USA). In still other embodiments, the nano- or releasing wound healing compositions to the wound bed for micro-beads are applied to the wound via a microneedle further healing. In some embodiments, a new (e.g., second, array. third, fourth, etc.) pad comprising impregnated wound heal (0169. C. Modification with Covalent Modifiers ing compositions as previously described is re-applied to the 0170 In some embodiments, the present invention pro wound wherein resides the biocompatible particles retained vides for the covalent immobilization of factors to the wound by the original application of the first pad/nanostructure. The bed. In some preferred embodiments, the covalent modifica application of the new pad, for example, serves not only to tion occurs via homobifunctional or heterobifunctional link impart fresh wound healing compositions to a wound but also ers. In some embodiments, the covalent modifiers are used to serves to recoat the biocompatible particles with fresh would directly modify the wound bed by covalently attaching the healing compositions that are released (e.g., either over a woundbed. In some embodiments, the covalent modifiers are short period of time or in a time-release manner) proximal to used to covalently attach 1, 2, 3, 4 or more different wound the wound bed. active agents to the wound bed. In some embodiments, the 0166 The present invention is not limited to a particular covalent modifiers are used to establish gradients of the 1, 2, mechanism. Indeed, an understanding of the mechanism is 3, 4 or more different wound active agents in a vertical or not necessary to practice the present invention. Nonetheless, horizontal plane with respect to the wound. In some embodi it is contemplated that the negatively charged macromolecu ments, the covalent modifiers are used in conjunction with the lar species and beads do not readily pass across cell mem polymer layers or beads describes above. In some embodi branes, and therefore serve as a non-toxic means of wound ments, the covalent modifies are used to attach wound active bed healing. As well, the size of the beads used determines the agents to the polymer layers or beads, while in other embodi nanostructure of the wound bed, thereby allowing for person ments, the covalent modifies are used to cross link the poly alized wound bed healing therapies. FIG. 6 provides an exem mers or beads. plary embodiment of the use of beads for woundbed healing. 0171 In some embodiments, the present invention pro However, the present invention is not limited by the size of vides methods of treatment, comprising providing a subject bead, the reactive groups found on the beads (e.g., carboxylic having a wound, at least one covalent modification agent and acid groups, thiol groups, amine groups, etc.), the cross at least one wound active agent, and contacting the wound linkers used, or the cytoactive factors and other wound heal with the at least one covalent modification agent and the at ing compositions that are associated with the beads. Indeed, a least one wound active agent under conditions such that the at myriad of combinations of compounds (e.g., Small molecule, least one wound active agent is covalently attached to the peptides, proteins, drugs, etc) for wound healing is contem wound. In some embodiments, the Subject is a human. In plated for use with beads applied to a wound bed. other embodiments, the Subject is a non-human vertebrate. In 0167. In some embodiments, the particles modulate a Some embodiments, the at least one covalent modification wound response following an application of an electric or agent is a homobifunctional cross-linker. In other embodi magnetic field to the wound bed. Accordingly, in some ments, the at least one covalent modification agent is a het embodiments, the particles are charged or magnetized so that erobifunctional cross-linker. For example, in some embodi the particles migrate or reorganize in an electric or magnetic ments, the homobifunctional cross-linker is an field. In other embodiments, the beads are dielectric so that N-hydroxysuccinimidyl ester (e.g., including, but not limited the beads experience dielectrophoresis in an AC electrical to, disuccinimidyl ester, dithiobis(Succinimidylpropionate), field. In other embodiments, the beads are magnetic and 3,3'-dithiobis(sulfosuccinimidylpropionate), disuccinimidyl migrate and/or transmit mechanical forces when exposed to a Suberate, bis(Sulfo Succinimidyl)suberate, disuccinimidyltar magnetic field. In other embodiments, the beads are charged tarate, disulfoSuccinimidyl tartarate, bis2-(Succinimidy so that mechanical forces are applied to the wound bed when loxycarbonyloxy)ethylsulfone, bis 2-(sulfoSuccinimi an electric field is applied, for example, by placing electrodes dooxycarbonyloxy)ethylsulfone, ethylene glycolbis on either side of a wound. (Succinimidylsuccinate), ethylene glycolbis 0168. In some embodiments, the nano- or micro-beads are (sulfoSuccinimidylsuccinate), disuccinimidyl glutarate, and applied to the wound by microfluidics printing, microstamp N,N'-disuccinimidylcarbonate). In some embodiments, the ing (U.S. Pat. Nos. 5,512,131 and 5,731,152, both of which homobifunctional cross-linker is at a concentration between 1 are incorporated by reference herein in their entirety), or nanomolar and 10 millimolar. In some preferred embodi microcontact printing (PCT Publication WO 96/29629, ments, the homobifunctional cross-linker is at a concentra incorporated by reference herein in its entirety). In other tion between 10 micromolar and 1 millimolar. In other embodiments, the nano- or micro-beads are applied to the embodiments, the at least one covalent modification agent is wound via a pulse jet such as an inkjet printer. Examples of a heterobifunctional cross-linker (e.g., including, but not lim suitable inkjet printers for application of biological fluids ited to, N-Succinimidyl 3-(2-pyridyldithio)propionate. Suc include those described in U.S. Pat. No. 7,128,398, WO cinimidyl 6-(3-2-pyridyldithio-propionamido)hexanoate, 95/25116 and WO 98/41531, each of which is incorporated sulfosuccinimidyl 6-(3'-2-pyridyldithio-propionamido) by reference in its entirety. In other embodiments, the nano hexanoate, succinimidyloxycarbonyl-O-methyl-O-(2-py or micro-beads are applied by drop dispensers such as the tip ridyldithio)toluene, sulfosuccinimidyl-6-O-methyl-O-(2- of a pin or in an open capillary tube and, touch the pin or pyridyldithio)toluamidohexanoate, succinimidyl 4-(N- capillary tube to the surface of the substrate. Such a procedure maleimidomethyl)cyclohexane-1-carboxylate, US 2011/O 1892.87 A1 Aug. 4, 2011 sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- 0.174. The present invention also provides kits for treating carboxylate, m-maleimidobenzoyl-N-hydroxySuccinimide a subject having a wound, comprising at least one covalent ester, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide modification agent, at least one wound active agent, and ester, N-Succinimidyl(4-iodoacetyl)aminobenzoate, Sulfo instructions for using the kit to covalently link the at least one Succinimidyl(4-iodoacetyl)aminobenzoate, Succinimidyl-4- wound active agent to the wound. In some embodiments, the (p-maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p-male at least one covalent modification agentis a homobifunctional imidophenyl)butyrate, N-(y-maleimidobutyryloxy) cross-linker, heterobifunctional crosslinker, or photoactivat Succinimide ester, N-(y-maleimidobutyryloxy) able crosslinker as described above. In some embodiments, SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) the at least one wound active agent includes, but is not limited hexanoate, Succinimidyl 6-(((6(4-iodoacetyl)amino) to wound active agents described in detail below. hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) 0.175. The present invention also provides a kit for treating amino)methyl)cyclohexane-1-carboxylate, Succinimidyl a subject having a wound, comprising at least one covalent 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) modification agent, at least one wound active agent, and amino)-hexanoate, and p-nitrophenyl iodoacetate). In some instructions for using the kit to covalently link the at least one embodiments, the heterobifunctional cross-linker is modified wound active agent to the wound. In some embodiments, the with functional groups, rendering it soluble in aqueous sol at least one covalent modification agentis a homobifunctional vents for delivery as an aqueous solution. Furthermore, in cross-linker. In some embodiments, the homobifunctional Some embodiments, the aqueous solution contains additives cross-linker is an N-hydroxySuccinimidyl ester (e.g., includ (e.g., including, but not limited to, Surfactants and block ing, but not limited to, disuccinimidyl ester, dithiobis(Succin copolymers). In other embodiments, a multiplicity of hetero imidylpropionate), 3,3'-dithiobis(sulfosuccinimidylpropi bifunctional cross-linkers can be attached to a molecule, onate), disuccinimidyl Suberate, bis(SulfoSuccinimidyl) polymer or particle to serve as the cross-linking agent. In Suberate, disuccinimidyl tartarate, disulfo Succinimidyl other embodiments, the heterobifunctional cross-linker is tartarate, bis 2-(Succinimidyloxycarbonyloxy)ethylsulfone, dissolved in an organic solvent (e.g., including, but not lim bis(2-(sulfosuccinimidooxycarbonyloxy)ethylsulfone, eth ited to, dimethyl sulfoxide). In some embodiments, the at ylene glycolbis(Succinimidylsuccinate), ethylene glycolbis least one wound active agent includes, but is not limited to, (sulfoSuccinimidylsuccinate), disuccinimidyl glutarate, and trophic factors, extracellular matrices, enzymes, enzyme N,N'-disuccinimidylcarbonate). In some embodiments, the at inhibitors, defensins, polypeptides, anti-infective agents (in least one covalent modification agent is a heterobifunctional cluding but not limited to silver (e.g., ionic silver, elemental cross-linker (e.g., including, but not limited to, N-succinim silver, silver nanoparticles)), buffering agents, vitamins and idyl 3-(2-pyridyldithio)propionate, succinimidyl 6-(3-2-py minerals, analgesics, anticoagulants, coagulation factors, ridyldithio-propionamido)hexanoate, SulfoSuccinimidyl anti-inflammatory agents, vasoconstrictors, vasodilators, 6-(3'-2-pyridyldithio-propionamido)hexanoate. Succinim diuretics, and anti-cancer agents. In some embodiments, the idyloxycarbonyl-O-methyl-O-(2-pyridyldithio)toluene, Sul at least one wound active agent contains one or more free-SH fosuccinimidyl-6-O-methyl-O-(2-pyridyldithio)toluamido groups. hexanoate, Succinimidyl 4-(N-maleimidomethyl) 0172. In some embodiments, the covalent modifier is a cyclohexane-1-carboxylate, SulfoSuccinimidyl 4-(N- photoactivatable crosslinker. Suitable photoactivatable maleimidomethyl)cyclohexane-1-carboxylate, crosslinkers include, but are not limited to, aryl azide N-((2- m-maleimidobenzoyl-N-hydroxysuccinimide ester, m-male pyridyldithio)ethyl)-4-azidosalicylamide, 4-azido-2,3,5,6- imidobenzoyl-N-hydroxy-sulfosuccinimide ester, N-succin tetrafluorobenzoic acid, Succinimidyl ester, 4-azido-2,3,5,6- imidyl(4-iodoacetyl)aminobenzoate, Sulfo-Succinimidyl(4- tetrafluorobenzoic acid, STP ester, benzophenone iodoacetyl)aminobenzoate, Succinimidyl-4-(p- maleimide, Succinimidyl ester of 4-benzoylbenzoic acid, maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p- N-5-Azido-2-Nitrobenzoyloxysuccinimide, N-Hydroxysul maleimidophenyl)butyrate, N-(y-maleimidobutyryloxy) fosuccinimidyl-4-azidobenzoate, N-Hydroxysuccinimidyl Succinimide ester, N-(y-maleimidobutyryloxy) 4-azidosalicylic acid, and (4-p-AZidosalicylamidobuty SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) lamine). hexanoate, Succinimidyl 6-(6-(((4-iodoacetyl)amino) 0173 Referring again to FIG. 2, spatial heterogeneity is hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) depicted in a wound by shapes 720 which depict different amino)methyl)cyclohexane-1-carboxylate, Succinimidyl chemical moieties found in a wound beds Such as amine 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) groups and Sulfhydryl groups. Typical areas in a wound will amino)-hexanoate, and p-nitrophenyl iodoacetate). In some be amine and or sulfhydryl-rich. In some embodiments, a embodiments, the at least one wound active agent includes, crosslinker (e.g., 230 in FIG. 2) comprising an activated acid but is not limited to, trophic factors (including polypetide is used to react with amine groups, for example nFIS (n-hy growth factors, neuropeptides, neurotrophins, extracellular droxysuccimydal) and a second crosslinker (e.g., 235 in FIG. matrices and their individual native constituents (exemplified 2) comprising a malemide is used to react with Sulfhydryl by but not limited to laminin, fibronectin, vitronectin, col groups. In some embodiments, the malimide and activated lagens, also select amino acid sequences found in these pro acid are conjugated to an alkyne so that a uniform surface is teins known to promote cell behaviors favorable to wound presented (e.g., 240 in FIG. 2 where X represents an alkyne healing e.g. integrin binding sequences exemplified by but group). From this point, widely available click chemistries not limited to RGD, EILDV,VCAM-1 and their recombined can be used to add desired wound active agents to the modi or synthetic analogs, enzymes, enzyme inhibitors, polypep fied surface of the wound. In some embodiments, the wound tides, antimicrobial peptides (exemplified by but not limited modifying agentis an azide conjugate or otherwise comprises to defensins, magaignins, cathelocidins, bactenicin) anti-in an azide group and is reacted with the alkyne groups dis fective agents (including but not limited to silver (e.g., ionic played on the wound bed in a Huisgen Cycloaddition. silver, elemental silver, silver nanoparticles)), buffering US 2011/O 1892.87 A1 Aug. 4, 2011 24 agents, vitamins and minerals, compounds that promote gen enzymatic degradation. The linkage may be one that is Sus eration/stabilization of nitic oxide, energy sources for cells, ceptible to degradation at a certain pH, for example. analgesics, anticoagulants, coagulation factors, anti-inflam 0180. In some embodiments, the one or more wound matory agents, vasoconstrictors, vasodilators, diuretics, and active agents are applied to form a gradient with respect to the anti-cancer agents. In other embodiments the kits include wound modifying agent. In general, the gradients present a small interfering RNAs (siRNAs-also referred to as micro higher contraction of wound active agent at one or more first RNAs) that are capable of promoting cellular behaviors con desired locations in the wound following application of the ducive to the wound healing process. In other embodiments, wound modifying agent to the wound and a lower concentra the kits include compounds that promote/stabilize a favorable tion of wound active agent at one or second location in the pH, osmotic environment, Surface energy, Surface charge, wound following application of the wound modifying agent Surface functionalities that enhance galvano and magneto to the wound. For example, the concentrations of the wound positive effects on wound healing, or balance of MMP/other active agents are layered in a wound bed in a gradient such peptidase/protease activity (i.e. inhibitors/promoters). that higher concentrations of a particular composition is greater proximal to the wound bed than distal to the wound (0176 II. Wound Active Agents bed in a vertical fashion. The converse, where concentrations 0177. In some embodiments, wound active agents are of compositions is greater distal to the woundbed than proxi delivered to a wound bed or incorporated into a wound bed mal, is also contemplated. Concentration of compositions in using the systems described above. In some embodiments, the a woundbed wherein a horizontal gradient is deposited is also wound active agent is bound covalently or non-covalently contemplated. Topographical gradients are also contem with the polyelectrolyte layer, bead, non-covalent modifier, plated, wherein compositions are deposited Such that the etc. The present invention is not limited to a particular mecha concentrations of compositions in a wound bed or on a bio nism by which the wound active agent binds to the polyelec compatible particle follow the topography of the substrate, trolyte layer, bead, non-covalent modifier, etc. for example, a higher concentration of compositions is depos 0178. In some embodiments, the polyelectrolyte layer, ited in the valleys of undulations of an exemplary substrate beads, or non-covalent modifier may function as a drug deliv compared to the peaks of the undulations. ery scaffold to deliver one or more wound active agents to the 0181. In some embodiments, the gradient comprises a wound. Wound active agents that may be desirable to deliver higher concentration of the wound active agent in the center include, but are not limited to, trophic factors, extracellular of the wound modifying agent which transitions to a lower matrices (ECMs), ECM fragments or synthetic constructs, concentration of the wound active agent away from the center enzymes, enzyme inhibitors, defensins, polypeptides, anti of the wound modifying agent. Accordingly, when the wound infective agents (including antimicrobials, antivirals and anti modifying agent is applied to a wound, the gradient results in fungals), buffering agents, vitamins and minerals, analgesics, a higher concentration of wound active agent in the center of anticoagulants, coagulation factors, anti-inflammatory the wound and a lower concentration of wound active agent as agents, vasoconstrictors, vasodilators, diuretics, and anti one moves to the periphery of the wound. In some embodi cancer agents. In addition, would active agents include iodine ments, the gradient comprises a lower concentration of the based antimicrobials such as PVP-iodine; selenium based wound active agent in the center of the wound modifying antimicrobials such as 7-aZabenzisoselenazol-3(2H)-ones, agent which transitions to a higher concentration of the Selenium disulfide, and selenides; and silver based antimicro wound active agent away from the center of the wound modi bials (e.g., silver Sulfadiazine, ionic silver, elemental silver, fying agent. Accordingly, the gradient results in a lower con silver nanoparticles)). With respect to selenides, with the use centration of wound active agent in the center of the wound of standard and variations of typical protein and carbohydrate and a higher concentration of wound active agent as one attachment chemistries, carboxyl and amino containing moves to the periphery of the wound. If two or more wound Selenides may be routinely attached to many polymers, pep active agents are utilized, they can be presented as similar tides, antibodies, steroids and drugs. Polymers and other mol gradients or the gradients can be varied so that the concentra ecules with attached selenides generate Superoxide in a dose tions of the two or more wound active agents vary across the dependent manner in biological Solutions, in cells or attached wound. The gradients of high or low concentration can be any to insoluble matrixes such as silicones. shape. Such as circular, square, rectangular, oval, oblong, etc. 0179 A wide variety of wound active agents can be incor so that the matrix and gradient can conform to a variety of porated into the polyelectrolyte layer, beads, or covalent or wound shapes. For example, for long, incision type wound, non-covalent modifier. The present invention is not limited to the gradient may be centered on a longitudinal axis that a particular mechanism by which one or more wound active extends along the length of the wound and can be centered on agents are released from the polyelectrolyte layer, beads, or the wound. As another example, the gradient can be circular covalent or non-covalent modifier into the wound. Indeed, an or oval-shaped for application to open type wounds, burns, understanding of the mechanism is not necessary to practice Sores and ulcers that are roughly circular or oval. In other the present invention. Nonetheless, in some embodiments, embodiments, the gradients comprise a series of features the present invention contemplates release of the one or more arranged in a pattern. For example, the gradients can form a incorporated agents from the polyelectrolyte layer, beads, or series of stripes or high and low concentrations of one or more non-covalent modifier to the wound by diffusion from the wound active agents along a longitudinal axis of the matrix. polyelectrolyte layer. In other embodiments, the one or more Alternatively, the gradients can form a checkerboard pattern, wound active agents may be released from the polyelectrolyte array, concentric circles, overlapping circles or oval, etc. layer, beads, or non-covalent modifier over time or in 0182. The present invention contemplates delivery of a response to an environmental condition. The one or more wide variety of wound active agents to the wound. In some wound active agents may be attached by a degradable linkage, embodiments, the present invention provides the delivery of Such as a linkage Susceptible to degradation via hydrolysis or trophic factors, including, but not limited to, agrin, amphi US 2011/O 1892.87 A1 Aug. 4, 2011
regulin, artemin, cardiotrophin-1, epidermal growth factors matrix, and polymeric matrices. In some embodiments, the including EGF; fibroblast growth factors (e.g., FGF-1, FGF wound active agents are integrin binding sequences exempli 2, FGF-3, FGF-4, FGF-5, FGF-6, and FGF-7); LIF, CSF-1, fied by, but not limited to RGD, EILDV, VCAM-1 and their recombined or synthetic analogs, enzymes, enzyme inhibi CSF-2, CSF-3, erythropoietin, endothelial cell growth factors tors, and polypeptides. including ECGF; FGF- and ECGF-related growth factors 0184. In some embodiments, the present invention pro (e.g., endothelial cell stimulating angiogenesis factor, tumor vides the delivery of enzymes, including, but not limited to, angiogenesis factor, retina-derived growth factor (RDGF). exopeptidases and endopeptidases (also known as proteases vascular endothelium growth factor (VEGF), brain-derived and proteinases), including but not limited to the serine pro growth factors (BDGF-A and B), astroglial growth factors teinases chymotrypsin, trypsin, elastase, and kallikrein, bac (AGF 1 and 2), omentum-derived growth factor, fibroblast terial enzymes, the cysteine proteases papain, actinin, brome stimulating factor (FSF), and embryonal carcinoma-derived lain, cathepsins, cytosolic calpains, parasitic proteases, growth factor (ECDGF)); neurotrophic growth factors (e.g. aspartic proteinases, the pepsin family of proteases pepsin nerve growth factors (NGFs), neurturin, brain-derived neu and chymosin, lysosomal cathepsins D, renin, fungal pro rotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, teases, the viral proteases, AIDS virus retropepsin, and the and ciliary neurotrophic factor (CNTF)); glial growth factors metalloproteinases (MMPs), collagenases, Maggott enzyme, (e.g., GGF-I, GGF-II, GGF-III, glia maturation factor MMP1, MMP2, MMP8, MMP13, gelatinases, MMP2, (GMF), and glial-derived neurotrophic factor (GDNF)); liver MMP9, MMP3, MMP7, MMP10, MMP11, and MMP12. growth factors (e.g., hepatopoietin A, hepatopoietin B, and 0185. In some embodiments, the present invention pro hepatocyte growth factors including HGF); prostate growth vides the delivery of enzyme inhibitors, including, but not factors including prostate-derived growth factors (PGFs); limited to captopril, thiorphan, phosphoramidon, teprotide, mammary growth factors including mammary-derived protease and proteinase inhibitors, metalloproteinase inhibi tors and exopeptidase inhibitors. In some embodiments, the growth factor 1 (MDGF-1) and mammary tumor-derived fac present invention provides the delivery of defensins, includ tor (MTGF); heart growth factors including nonmyocyte ing, but not limited to, alpha-defensins HNP 1,2,3 and 4, and derived growth factor (NMDGF); melanocyte growth factors beta-defensins HBD-1 and HBD-2. including melanocyte-stimulating hormone (MSH) and 0186. In some embodiments, the present invention pro melanoma growth-stimulating activity (MGSA); angiogenic vides the delivery of polypeptides, including, but not limited factors (e.g., angiogenin, angiotropin, platelet-derived ECGF. to, fibronectin, serotonin, PAF, PDEGF, TNFa. ILLIL6, IGF, VEGF, and pleiotrophin); transforming growth factors IGF-1, IGF-2, IL-1, PDGF, FGF, KGF, VEGF, bradykinin, including TGF-C. and TGF-?3: TGF-like growth factors (e.g., prothymosin-alpha, and thymosin-alphal. In some embodi TGF-beta, TGF-beta, TGF-beta GDF-1, CDGF, tumor ments, the present invention provides the delivery of antimi derived TGF-like factors, ND-TGF, and human epithelial crobials, including, but not limited to, magainin (e.g., magai transforming factor); regulatory peptides with growth factor nin I, magainin II, Xenopsin, Xenopsin precursor fragment, like properties (e.g., bombesin and bombesin-like peptides caerulein precursor fragment), magainin I and II analogs ranatensin and litorin, angiotensin, endothelin, atrial natri (e.g., PGLa, magainin A, magainin G, pexiganin, Z-12, pexi uretic factor, vasoactive intestinal peptide, and bradykinin); gainin acetate, D35, MSI-78A, MGO (K10E, K11E, FI2W platelet-derived growth factors including PDGF-A, PDGF-B, magainin 2), MG2+(K10E, F12W-magainin-2), MG4+ and PDGF-AB; neuropeptides (e.g., substance P. calcitonin (F12W-magainin2), MG6+ (f12W, E19Q-magainin2 amide), gene-regulated peptide (CGRP), and neuropeptide Y); neu MSI-238, reversed magainin II analogs (e.g., 53D. 87-ISM, rotransmitters and their analogs including norepinephrine, and A87-ISM), Ala-magainin II amide, magainin II amide), acetylcholine and carbachol; hedgehog, heregulin/neuregu cecropin Pl, cecropinA, cecropin B, indolicidin, nisin, ranal lin, IL-1, osteoclast-activating factor (OAF), lymphocyte-ac exin, lactoferricin B, poly-L-lysine, cecropin A (1-8)-magai tivating factor (LAF), hepatocyte-stimulating factor (HSF), nin II (1-12), cecropin A (1-8)-melittin (1-12), CA(1-13)-MA B-cell-activating factor (BAF), tumor inhibitory factor 2 (1-13), CA(1-13)-ME(1-13), gramicidin, gramicidin A, (TIF-2), keratinocyte-derived T-cell growth factor (KD gramicidin D. gramicidin S, alamethicin, protegrin, histatin, TCGF), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, dermaseptin, lentivirus amphipathic peptide or analog, para IL-11, stromal cell-derived cytokine (SCDC), IL-12, IL-13, sin I, lycotoxin I or II, globomycin, gramicidin S, Surfactin, IL-14, IL-15, insulin, insulin-like growth factors including ralinomycin, Valinomycin, polymyxin B, PM2 ((+/-) 1-(4- IGF-1, IGF-2, and IGF-BP; interferons including INF-alpha, aminobutyl)-6-benzylindane), PM2c ((+/-)-6-benzyl-1-(3- INF-beta, and INF-gamma; leptin, midkine, tumor necrosis carboxypropyl)indane), PM3 ((+/-)1-benzyl-6-(4-aminobu factors (TNF-alpha and beta), metrins, saposins, semaphorins, tyl)indane), tachyplesin, buforin I or II, misgurin, melittin, somatrem, somatropin, stem cell factor, VVGF, bone mor PR-39, PR-26, 9-phenylnonylamine, (KLAKKLA)n, phogenetic proteins (BMPs), adhesion molecules, other (KLAKLAK)n, where n=1, 2, or 3, (KALKALK)3, cytokines, heparin-binding growth factors, and tyrosine KLGKKLG)n, and KAAKKAA)n, wherein N=1, 2, or 3, kinase receptor ligands. In some embodiments, the wound paradaxin, Bac 5. Bac 7, ceratoxin, mdelin 1 and 5, bombin active agent is a peptide such as AcEEED, which is the N like peptides, PGQ, cathelicidin, HD-5, Oabac5alpha, terminal peptide for alpha Smooth muscle actin and has been ChBac5, SMAP-29, Bac7.5, lactoferrin, granulysin, thionin, shown to inhibit contractile properties of myofibroblasts. hevein and knottin-like peptides, MPG 1, 1bAMP, snakin, 0183 In some embodiments, the present invention pro lipid transfer proteins, and plant defensins. Exemplary vides the delivery of ECMs, including, but not limited to sequences for the above compounds are provided in Table 1. native constructs, fragments of native constructs and Syn In some embodiments, the antimicrobial peptides are synthe thetic analogs of extracellular matrix proteins, reconstituted sized from L-amino acids, while in other embodiments, the basement membrane-like complexes derived from eukaryotic peptides are synthesized from, or comprise, D-amino acids. cell lines, collagens, fibronectin, laminin, VCAM-1, Vit Additional antimicrobial polypeptides of use in the present ronectin and gelatin, a bacterial extracellular matrix, a gel invention are listed in FIG. 7.
US 2011/O 1892.87 A1 Aug. 4, 2011 27
TABLE 1 - continued Antimicrobial Peptides SEO ID NO: Name Organism Sequence
23 dermasept in 3 Phyllomedusa alwknmlkgigklagkaalgavkklvgaes sauvagei
24 misguirin Misgurnus rqrveel skifskkgaaarrrk anguillicaudatus
25 melittin Apis mellifera gigavlkvlittglpalliswisrkkrqq
26 pardaxin-1 Pardachirus gffalipki is splifktill savgsals ssgege pavoninus
27 pardaxin-2 Pardachirus gffalipki is spifktill savgsals sisggge pavoninus
28 bactenecin 5 Bos taurus metgrasl slgrcslwllllgilvlp sasaqal Syreavlr precursor avddfners seanlyrlleldptpnddldpgtrkpvs frv ketc.cprtscqpleq.cdfkenglivkgcvgtvtldpsindof dincinelq Svirfropirrppirppfyppfropirppifpp irppfroplgpfpgrr
29 bactenecin Bos taurus metprasl slgrwslw1111glalp Sasaqal Syreavlr precursor avddlineqssepniyrlleldoppgddedpdspkirvis frv ketv.csirttggppeqcdfkengllkrceg tvtildovirginf ditcnnhosiritkopwappgaarl Crivvirvor
ceratotoxin A Ceratitis sigsalikkalpvakkigkialpiakaalp capitata
31 ceratotoxin B Ceratitis sigsafikkalpvakkigkaalpiakaalp capitata
32 cathelicidin Homo sapiens mktoringhslgrwslvillllgilvmplaiiaqvlsykeavil antimicrobial raidgingrsscanlyrill dildprotmdgdpdtpkp vsft peptide vketv.cprttgospedcofkkdglvkrcmgtvtlinqargs fails cakdnkrfallgdiffrkskekigkefkrivdrikdf lirnlvprites
33 myeloid Equus cabalius metorntrol grwsplllllgilvippattgal Sykeavlr cathelicidin 3 avdglingrsscenllyrilleldplpkgdkos dtpkp vsfmv ketv.cprimkotpeg.cdfkenglivkgcvgtvildpvkdyf dascolepqrvkrfhsvg sligrhagmirdkseatrhgiri itrpkilllas
34 myeloid Bos taurus metgrasl slgrwslwllllglalp Sasaqal Syreavlr antimicrobial avdolneks seanlyrlleldpppkeddenpnipkp vs fr peptide BMAP-28 vketv.cprtscq speqcdfkengllkecvgtvtldovg Sn folit cavpqsvgglrslgrkillrawkkygpiivpiirig
35 myeloid Equus cabalius metorntrol grwsplllllgilvippattgal Sykeavlr cathelicidin 1 avdglingrsscenllyrilleldplpkgdkos dtpkp vsfmv ketv.cprimkotpeg.cdfkenglivkgcvgtvilgpvkdhf divscgeporvikrfgrlaks flirmirillprrkill as
36 SMAP 29 Ovis airies metgrasl slgrcslwllllglalp Sasaqvil Syreavlr aadqlneks seanlyrlleldpppkodden Snipkp vs fr vketv.cprtscopaedic difkengllkecvgtvtldovrnn foliticaepcis Virglrrlgrkiahgvkkygptvlriiriag
37 Bos taurus crivvivor
38 Homo sapiens acycripaciagerrygtciyggirl wafcc
39 Homo sapiens cycripacilagerrygt ciyggirlwafcc
4 O Homo sapiens dcycripaciagerrygtciyggirl wafcc
41 Homo sapiens vcscrlvificirrtelrvgncliggvsftycotrv
42 Oryctolagus vvcacirralclprerragfcrirgrihplc cirr cliniculus US 2011/O 1892.87 A1 Aug. 4, 2011 28
TABLE 1 - continued Antimicrobial Peptides SEO ID NO: Name Organism Sequence
43 NP-2 Oryctolagus vvcacirralclplerragfcrirgrihplc cirr cliniculus
44 Oryctolagus gicacirrirfcpnserfs gy crvngary virc.csirr cliniculus
45 Oryctolagus grovcirkollic syrerrigdckirgvrfpfcc.pr cliniculus
46 NP-4 Oryctolagus vSct cirrfscgfgerasgs ctvnggvrhtlccrr cliniculus
47 NP-5 Oryctolagus vfict crgflicgsgerasgs cting vrhtlic cirr cliniculus
48 RatP-1 Rattus vtcy cirrtrcgfrerilsgacgyrgriyrlic cr norvegicus
49 Rat-NP-3 Rattus c scrys scrif gerllsgacrlingriyrlicc norvegicus
SO Rat-NP-4 Rattus act crigacvsgerltgacglingriyl.ccr norvegicus
51 GPNP Guinea pig rrcict trtcrfpyrrligt cifonrvytfcc
52 beta defensin-3 Homo sapiens mrihyll fallfliflvpvpghggiintlokyycrvirggrd avls clipkeeqigkcstrogrkccrrkk.
53 theta defensin-1 Macaca mulatta rcictrgfcirclcrrgvc
54 defensin CUA1 Helianthus mkssmkmfaallilvvmcillanemggplvveartcesqshk S fkgtclsatncanvchserfsggkcrgfrirrcfctthc
55 defensin SD2 Helianthus mkssmkmfaallilvvmcillanemggplvveartcesqshk S fkgtclsatncanvchserfsggkcrgfrirrcfctthc
56 neutrophil Macaca mulatta acycripaclagerrygtcfymgirv Wafcc defensin 2
st 4 KDA defensin Androctonus gfgcpfnggachrhcrSirirriggycaglfkot ct cyr australis hector
58 defensin Mytilus gfgCpnny qchrhcksipgrcggycggxhrlrict Cyrc galloprovincia lis
59 defensin AMP1 Heuchera digvklcdvpsgtwsghcgs sskosqqckdrehfayggach sanguinea yofpsvkcfckroc
60 defensin AMP1 Clitoria nlcerasltwtgnogntghcdtocrinWesakhgachkirgin tellatea wkcfcyfnc
61 cysteine-rich Mus musculus mkklvillfalvillafovoads ignt deetkteeqpgekdq cryptdin-1 avsvsfgdpggs algolaalgwgrrcpdcpropscps cpro homolog proprickcnpk
62 beta-defensin-9 Bos taurus qgvrnfvt crinirgfcvpircpghrirgigt clgpdikccr
63 beta-defensin-7 Bos taurus qgvrnfvt crinirgfcvpircpghrirgigt clgprikccr
64 beta-defensin- 6 Bos taurus qgvrnhvt criyggfcvpircpgrtroigt cfgrpvkccr W
65 beta-defensin-5 Bos taurus qv Vrnpos crWnmgvcipiscpgnmirqigt cfgprvpccr
66 beta-defensin- 4 Bos taurus qrvrnpos crWnmgvcipflcrvgmroigt cfgprvpccr
67 beta-defensin-3 Bos taurus qgvrnhvt crinirgfcvpircpgrtroigt cfgprikccr SW
68 beta-defensin-10 Bos taurus qgvir Sylscwgnirgicillnircpgrmroigt clap rvkccr
US 2011/O 1892.87 A1 Aug. 4, 2011 30
TABLE 1- Continued Antimicrobial Peptides SEO ID NO: Name Organism Sequence
92 neutrophil Mesocricetus cfckrpvcdsgetoigy crligntifyirl.ccrq defensin. 1 aliats
93 Gallinacin Gallus gallus grksdcfrkingficaflkcpyltlisgkcsirfhlcckriw 1-alpha
94 defensin Allomyrina vtcdlls feakgfaanhsil caahclaigrriggs cergvci dichotoma c 95 neutrophil Cavia porcelius rrcict trtcrfpyrrligt cifonrvytfcc cationic peptide 1
0187. In some embodiments, the present invention pro quine phosphate, trichomonocides, anthelmintics, atova vides the delivery of antimicrobials, including, but not limited quone, bacitracin, bacitracin/polymyxin b, gentamycin, to, loracarbef cephalexin, cefadroxil, cefixime, ceftibuten, neomycin/polymyxin/dexameth, neomycin Sulf dexameth, cefprozil, cefpodoxime, cephradine, cefuroxime, cefaclor, Sulfacetamide/prednisolone, Sulfacetamide/phenylephrine, neomycin/polymyxin/bacitracin, dicloxacillin, nitrofuran tobramycin sulfate/dexameth, bismuth tribromophenate, sil toin, nitrofurantoin macrocrystal, nitrofurantoin/nitrofuran ver ion compounds, silver nanoparticles, Zerovalent silver, mac, dirithromycin, gemifloxacin, amplicillin, gatifloxacin, multivalent silver, elemental silver, and silver containing penicillin V potassium, ciprofloxacin, enoxacin, amoxicillin, compounds such as silver Sulfadiazine and related com amoxicillin/clavulanate potassium, clarithromycin, levof pounds. loxacin, moxifloxacin, azithromycin, sparfloxacin, cefdinir, 0188 In some embodiments, the present invention pro ofloxacin, trovafloxacin, lomefloxacin, methenamine, eryth vides the delivery of antivirals, including, but not limited to, romycin, norfloxacin, clindamycin/benzoyl peroxide, quinu amantadine, acyclovir, foscarnet, indinavir, ribavirin, enfu pristin?dalfopristin, doxycycline, amikacin Sulfate, Vancomy Virtide, emtricitabine, lamivudine, abacavir sulfate, cin, kanamycin, netilmicin, streptomycin, tobramycin fomivirsen, Valacyclovir, tenofovir, cidofovir, atazanavir, Sulfate, gentamicin Sulfate, tetracyclines, framycetin, amprenavir, delavirdine mesylate, famciclovir, adefovir, minocycline, nalidixic acid, demeclocycline, trimethoprim, didanosine, efavirenz, trifluridine, inidinavir, lamivudine, miconazole, colistimethate, piperacillin sodium/taZobactam Vidarabine, lopinavir/ritonavir, ganciclovir, Zanamivir, aba Sodium, paromomycin, colistin/neomycin/hydrocortisone, cavir/lamivudine/zidovudine, lamivudine/zidovudine, nelfi amebicides, Sulfisoxazole, pentamidine, Sulfadiazine, clinda navir, nelfinavir mesylate, nevirapine, ritonavir, saquinavir, mycin phosphate, metronidazole, oxacillin Sodium, nafcillin saquinavir meSylate, rimantadine, stavudine, docosanol, Zal Sodium, Vancomycin hydrochloride, clindamycin, cefo citabine, idoxuridine, Zidovudine, Zidovudine/didanosine, taxime sodium, co-trimoxazole, ticarcillin disodium, piper Valganciclovir, penciclovir, lamivudine, and oseltamivir. acillin sodium, ticarcillin disodium/clavulanate potassium, 0189 In some embodiments, the present invention pro neomycin, daptomycin, cefazolin Sodium, cefoxitin Sodium, vides the delivery of antifungals, including, but not limited to, ceftizoxime sodium, penicillin G potassium and Sodium, amphotericin B, nystatin, nystatin/triamcinolone, itracona ceftriaxone sodium, ceftazidime, imipenem/cilastatin Zole, ketoconazole, miconazole, Sulconazole, clotrimazole, Sodium, aztreonam, cinoxacin, erythromycin/sulfisoxazole, clotrimazole/betamethasone, enilconazole, econazole, oxi cefotetan disodium, ampicillin Sodium/sulbactam Sodium, conazole, tioconazole, terconazole, butoconazole, thiabenda cefoperaZone sodium, cefamandole nafate, gentamicin, Zole, flucytosine, butenafine, ciclopiroX, haloprogin, nafti Sulfisoxazole/phenaZopyridine, tobramycin, lincomycin, fine, tolnaftate, natamycin, undecylenic acid, mafenide, neomycin/polymyxin B/gramicidin, clindamycin hydrochlo dapsone, clioquinol, clioquinol/hydrocortisone, potassium ride, lanSoprazole?clarithromycin/amoxicillin, alatrofloxa iodide, silver Sulfadiazine, gentian violet, carbol-fuchsin, cin, linezolid, bismuth subsalicylate/metronidazole/tetracy cillofungin, Sertaconazole, Voriconazole, fluconazole, terbin cline, erythromycin/benzoyl peroxide, mupirocin, afine, caspofungin, other topical azole drugs, and griseoful fosfomycin, pentamidine isethionate, imipenem/cilastatin, W1 troleandomycin, gatifloxacin, chloramphenicol, cycloserine, 0190. In some embodiments, the present invention pro neomycin/polymyxin B/hydrocortisone, ertapenem, mero vides the use and delivery of buffering agents, including, but penem, cephalosporins, fluconazole, cefepime, Sulfamethox not limited to, Maleic acid, Phosphoric acid, Glycine, Chlo azole, Sulfamethoxazoleftrimethoprim, neomycin/poly roacetic acid, Formic acid, Benzoic acid, Acetic acid, Pyri myxin B. penicillins, rifampin/isoniazid, erythromycin dine, Piperazine, MES, Bis-tris, Carbonate, ACES, ADA estolate, erythromycin ethylsuccinate, erythromycin Stearate, MOPSO, PIPES, Phosphoric acid, BES, MOPS, TES, ampicillin trihydrate, amplicillin/probenecid, Sulfasalazine, HEPES, DIPSO, TAPSO, Triethanolamine, HEPSO, Tris, Sulfanilamide, Sodium sulfacetamide, dapsone, doxycycline Tricine, Bicine, TAPS, Borate, Ammonia, CHES, Ethanola hyclate, trimenthoprim/sulfa, methenamine mandelate, plas mine, CAPSO, Glycine, Carbonate, CAPS, Methylamine, modicides, pyrimethamine, hydroxychloroquine, chloro Piperidine, and Phosphoric acid. US 2011/O 1892.87 A1 Aug. 4, 2011
0191 In some embodiments, the present invention pro Artofen, Dolgit, Artril, Dolocyl, Bloom, Donjust, Bluton, vides the delivery of vitamins and minerals, including, but not Easifon, Ebufac, Emflam, Emodin, Fenbid, Fenspan, Focus, limited to, Vitamin A, Carotenoids, Vitamin D. Vitamin E, Ibosure, Ibufen, Ibufug, Ibugen, Ibumetin, Ibupirac, Imbun, Vitamin K, Vitamin C/ascorbic acid, Bl/thiamin, B2/ribofla Inabrin, Inflam, Irfen, Librofen, Limidon, Lopane, Mynose vin, B3/niacin, B5/pantothenic acid, B6/pyridoxine, B12/co din, Napacetin, Nobafon, Nobgen, Novogent, Novoprofen, balamin, Biotin, Calcium, Magnesium, Phosphorus, Sodium, Nurofen, Optifen, Paduden, Paxofen, Perofen, Proartinal, Chloride, Potassium, Boron, Chromium, Copper, Iodine, Prontalgin, Q-Profen, Relcofen, Remofen, Roidenin, Seclo Iron, Manganese, Selenium, and Zinc. din, Tarein, and Zofen), indomethacin (also known as 0.192 In some embodiments, the present invention pro Indameth, Indocin, Amuno, Antalgin, Areumatin, Argilex, vides the delivery of analgesics, including, but not limited to, Artherexin, Arthrexin, Artrinovo, Bavilon, Bonidon, Bouty acetaminophen, anilleridine, acetylsalicylic acid, buprenor cin, Chrono-Indocid, Cidalgon, Confortid, Confortind, phine, butorphanol, fentanyl, fentanyl citrate, codeine, rofe Domecid. Durametacin, Elemetacin, Idicin, Imbrilon, Inacid, coxib, hydrocodone, hydromorphone, hydromorphone Indacin, Indecin, Indocap, Indocen, Indocid, Indoflex, hydrochloride, levorphanol, alfentanil hydrochloride, mep Indolag, Indolar, Indomed, Indomee, Indometacinum, eridine, meperidine hydrochloride, methadone, morphine, Indometicina, Indometin, Indovis, IndoX, IndoZu, Indrenin, nalbuphine, opium, levomethadyl, hyaluronate Sodium, Indylon, Inflazon, Inpan, Lauzit, Liometace, Metacen, Met Sufentanil citrate, capsaicin, tramadol, leflunomide, oxyc indon, Metocid, Mezolin, Mobilan, Novomethacin, Peralgon, odone, oxymorphone, celecoxib, pentazocine, pro Reflox, Rheumacid, Rheumacin, Salinac, Servindomet, poxyphene, benzocaine, lidocaine, dezocine, clonidine, Toshisan, and Vonum), ketoprofen (also known as Orudis, butalbital, phenobarbital, tetracaine, phenaZopyridine, Sul Alrheumat, Alrheumun, Alrhumat, Aneol, Arcental, Dexal, famethoxazole/phenazopyridine, and sulfisoxazole? Epatec, Fastum, Keduril, Kefenid, Keprofen, Ketofen, Ket phenaZopyridine. onal, Ketosolan, Kevadon, Mero, Naxal, Oruvail, Profenid, 0193 In some embodiments, the present invention pro Salient, Tofen, and Treosin), ketorolac (also known as Tora vides the delivery of anticoagulants, including, but not lim dol), meclofenamate (also known as Meclofen, Meclomen, ited to, coumarins, 1.3-indandione, anisindione, fonda and Movens), mefenamic acid (also known as Ponstel, parinux, heparin, lepirudin, antithrombin, warfarin, Alpain, Aprostal, Benostan, Bonabol, Coslan, Dysman, Dys enoxaparin, dipyridamole, dalteparin, ardeparin, nadroparin, pen, Ecopan, Lysalgo, Manic, Mefac, Mefic, Mefix, and tinzaparin. Parkemed, Pondex, Ponsfen, Ponstan, Ponstyl, Pontal, Ral 0194 In some embodiments, the present invention pro gec, and Youfenam), nabumetone (also known as Relafen), vides the delivery of coagulation factors, including, but not naproxen (also known as Naprosyn, AnaproX, Aleve, limited to, Factor I (fibrinogen), Factor II (prothrombin), Apranax, Apronax, Arthrisil, Artrixen, Artroxen, Bonyl, Con Factor III (thromboplastin, tissue factor), Factor IV (cal gex, DanaproX, Diocodal, Dysmenalgit, Femex, Flanax, cium), Factor V (labile factor), Factor VII (stable factor), Flexipen, Floginax, Gibixen, Headlon, Laraflex, Laser, Len Factor VIII (antihemophilic globulin, antihemophilic globu iartil, Nafasol, Naixan, Nalyxan, Napoton, Napren, Naprelan, lin, antihemophilic factor A), Factor IX (plasma thrombo Naprium, Naprius, Naprontag, NapruX, NapXen, Narma, plastin component, Christmas factor, antihemophilic factor Naxen, Naxid, Novonaprox, Nycopren, Patxen, Prexan, Pro B), Factor X (Stuart factor, Prower factor, Stuart-Prower fac dexin, Rahsen, Roxen, Saritilron, Sinartrin, Sinton, Sutony, tor), Factor XI (plasma thromboplastin antecedent, antihemo Synflex, Tohexen, Veradol, Vinsen, and Xenar), oxaprozin philic factor C), Factor XII (Hageman factor, surface factor, (also known as Daypro), piroXicam (also known as Feldene, contact factor), and Factor XIII (fibrin stabilizing factor, Algidol, Antiflog, Arpyrox, Atidem, Bestocam, Butacinon, fibrin stabilizing enzyme, fibri-nase). Desinflam, Dixonal, Doblexan, Dolonex, Feline, FelroX, Full 0.195. In some embodiments, the present invention pro din, Indene, Infeld, Inflamene, Lampoflex, Larapam, Medop vides the delivery of anti-inflammatory agents, including, but til, Novopirocam, Osteral, Pilox, Piraldene, Piram, Pirax, not limited to, non steroidal anti-inflammatory drugs Piricam, Pirocam, Pirocaps, Piroxan, Piroxedol, Piroxim, (NSAIDs) including diclofenac (also known as Voltaren, Abi Piton, Posidene, Pyroxy, Reucam, Rexicam, Riacen, Rosic, tren, Alvoran, Almiral. Alonpin, Anfenax, Artrites, Betaren, Sinalgico, Sotilen, Stopen, and Zunden), Sulindac (also Blesin, Bollabomin, Cataflam, Clofec, Clofen, Cordralan, known as Clinoril, Aflodac, Algocetil, Antribid, Arthridex, Curinflam, Diclomax, Diclosian, Dicsnal, Dilfenac, Arthrocine, Biflace, Citireuma, Clisundac, Imbaral, Lindak, Ecofenac, Hizemin, Inflamac, Inflanac, Klotaren, Lidonin, Lyndak, Mobilin, Reumofil, Sudac, Sulene, Sulic, Sulindal, Monoflam, Naboal, Oritaren, Remethan, Savismin, Silino, Suloril, and Sulreuma), tolmetin (also known as Tolectin, Staren, Tsudohmin, Voltarol, Voren, Voveran, and Vurdon), Donison, Midocil, Reutol, and Safitex), celecoxib (also diflunisal (also known as Dolobid, Adomal, Diflonid, Diflu known as Celebrex), meloxicam (also known as Mobic), rofe nil, Dolisal, Dolobis, Dolocid, Donobid, Dopanone, Dorbid, coxib (also known as Vioxx), Valdecoxib (also known as Dugodol, Flovacil, Fluniget, Fluodonil, Flustar, Ilacen, Noal Bextra), aspirin (also known as Anacin, AScriptin, Bayer, dol, Reuflos, and Unisal), etodolac (also known as Lodine), Bufferin, Ecotrin, and Excedrin) and steroidal anti-inflamma fenoprofen (also known as Nalfon, Fenoprex, Fenopron, tory drugs including cortisone, prednisone and dexametha Fepron, Nalgesic, and Progesic), flurbiprofen (also known as SO. Ansaid and Ocuflur), ibuprofen (also known as Rufen, Mot 0196. In some embodiments, the present invention pro rin, Aches-N-Pain, Advil, Nuprin, Dolgesic, Genpril, Halt vides the delivery of vasoconstrictors, including, but not lim ran, Ibifon, Ibren, Ibumed, Ibuprin, Ibupro-600, Ibuprohm, ited to, epinephrine (adrenaline, Susphrine), phenylephrine Ibu-Tab, Ibutex. Ifen, Medipren, Midol 200, Motrin-IB, hydrochloride (Neo-Synephrine), oxymetazoline hydrochlo Cramp End, Profen, Ro-Profen, Trendar, Alaxan, Brofen, ride (Afrin), norepinephrine (Levophed), and caffeine. Alfam, Brufen, Algofen, Brufort, Amersol, Bruzon, Andran, 0.197 In some embodiments, the present invention pro Buburone, Anflagen, Butacortelone, Apsifen, Deflem, vides the delivery of vasodilators, including, but not limited US 2011/O 1892.87 A1 Aug. 4, 2011 32 to, bosentan (Tracleer), epoprostenol (Flolan), treprostinil mus, tamoxifen, temozolomide, teniposide, testolactone, (Remodulin), sitaxsentan, nifedipine (Adalat, Procardia), thioguanine, thiotepa, topotecan, toremifene, toSitumomab, nicardipine (Cardene), Verapamil (Calan, Covera-HS, Isop trastuzumab, tretinoin, uracil mustard, valrubicin, vinblas tin, Verelan), diltiazem (Dilacor XR, Diltia XT, Tiamate, tine, Vincristine, Vinorelbine, and Zoledronate. Tiazac, Cardizem), isradipine (DynaCirc), nimodipine 0200. In other embodiments, the wound active agent is an (Nimotop), amlodipine (Norvasc), felodipine (Plendil), siRNA. The RNAi constructs of the present invention are nisoldipine (Sular), bepridil (Vascor), hydralazine (Apreso gene(s) that express RNAs that base pair to form a dsRNA line), minoxidil (Loniten), isosorbide dinitrate (Dilatrate-SR, RNA region. The RNAs may be a part of the same molecule Iso-Bid, Isonate, Isorbid, Isordil, Isotrate, Sorbitrate), isor or different molecules. In preferred embodiments, the RNAi bide mononitrate (IMDUR), prazosin (Minipress), cilostazol construct comprises a promoter operably linked to a nucleic (Pletal), treprostinil (Remodulin), cyclandelate, isoxSuprine acid sequence encoding two complementary sequences sepa (Vasodilan), nylidrin (Arlidin), nitrates (Deponit, Minitran, rated by a loop sequence. The complementary regions corre Nitro-Bid, Nitrodisc, Nitro-Dur. Nitrol, Transderm-Nitro), spond to a target RNA sequence separated by a loop benazepril (Lotensin), benazepril and hydrochlorothiazide sequence. When the RNAi construct is expressed, the (Lotensin HCT), captopril (Capoten), captopril and hydro complementary regions of the resulting RNA molecule pair chlorothiazide (Capozide), enalapril (Vasotec), enalapril and with one another to form a double stranded RNA region. The hydrochlorothiazide (Vaseretic), fosinopril (Monopril), lisi present invention is not limited to loop sequences of any nopril (Prinivil, Zestril), lisinopril and hydrochlorothiazide particular length. In some preferred embodiments, the loop (Prinzide, Zestoretic), moexipril (Univasc), moexipril and sequences range from about 4 to about 20 nucleotides in hydrochlorothiazide (Uniretic), perindopril (Aceon), length. In more preferred embodiments, the loop sequences quinapril (Accupril), quinapril and hydrochlorothiazide (Ac are from about 6 to about 12 nucleotides in length. In other curetic), ramipril (Altace), trandolapril (Mavik), papaverine preferred embodiments, the dsRNA regions are from about 19 (Cerespan, Genabid, Pavabid, Pavabid HP, Pavacels, Pavacot, to about 23 in length. Pavagen, Pavarine, Pavased, Pavatine, Pavatym, Paverolan). 0201 In other embodiments, the dsRNA is formed from 0198 In some embodiments, the present invention pro RNA transcribed from a vector as two separate stands. In vides the delivery of diuretics, including, but not limited to, other embodiments, the two strands of DNA used to form the acetazolamide (Diamox), dichlorphenamide (Daranide), dsRNA may belong to the same or two different duplexes in methazolamide (Neptazane), bendroflumethiazide (Nature which they each form with a DNA strand of at least partially tin), benzthiazide (Exna), chlorothiazide (Diuril), chlorthali complementary sequence. When the dsRNA is thus-pro done (Hygroton), hydrochlorothiazide (Esidrix, HydroDi duced, the DNA sequence to be transcribed is flanked by two uril, Microzide), hydroflumethiazide (Diucardin), promoters, one controlling the transcription of one of the indapamide (LoZol), methyclothiazide (Enduron), metola strands, and the other that of the complementary strand. These Zone (ZaroXolyn, Mykrox), polythiazide (Renese), quineth two promoters may be identical or different. In some embodi aZone (Hydromox), trichlormethiazide (Naqua), bumetanide ments, a DNA duplex provided at each end with a promoter (Bumex), ethacrynic acid (Edecrin), furosemide (Lasix), sequence can directly generate RNAS of defined length, and torsemide (Demadex), amiloride (Midamor), amiloride and which can join in pairs to form a dsRNA. See, e.g., U.S. Pat. hydrochlorothiazide (Moduretic), spironolactone (Aldac No. 5,795,715, incorporated herein by reference. RNA tone), spironolactone and hydrochlorothiazide (Aldactazide), duplex formation may be initiated either inside or outside the triamterene (Dyrenium), triamterene and hydrochlorothiaz cell. ide (Dyazide, Maxzide). 0202. It will be recognized that after processing the result 0199. In some embodiments, the present invention pro ing siRNA can comprise two blunt ends, one blunt end and vides the delivery of anti-cancer agents, including, but not one end with an overhang, or two ends with overhangs. In limited to, aldesleukin, alemtuzumab, alitretinoin, allopu Some embodiments, the end or ends with overhangs comprise rinol, altretamine, amifostine, anagrelide, anastrozole, an overhang of either one or two nucleotides. As a non arsenic trioxide, asparaginase, bexarotene, bicalutamide, limiting example, a siRNA of 23 nucleotides in length com bleomycin, buSulfan, calusterone, capecitabine, carboplatin, prises two 19 mers with a two nucleotide overhang at each carmustine, celecoxib, chlorambucil, cisplatin, cladribine, end. As another non-limiting example, a siRNA of 21 nucle cyclophosphamide, cytarabine, dacarbazine, dactinomycin, otides in length comprises two 19 mers with a single nucle darbepoetin alpha, daunorubicin, daunomycin, dexraZOxane, otide overhang at each end. As still another non-limiting docetaxel, doxorubicin, epoetin alpha, estramustine, etopo example, a siRNA of 22 nucleotides in length comprises two side, etoposide phosphate, exemestane, filgrastim, floXuri 22 mers with no overhangs at either end. dine, fludarabine, flutamide, fulvestrant, gemcitabine, gem 0203 Inhibition is sequence-specific in that nucleotide tuZumab ozogamicin, goserelin acetate, hydroxyurea, sequences corresponding to the duplex region of the RNA are ibritumomabtiuxetan, idarubicin, ifosfamide, imatinib mesy targeted for genetic inhibition. RNA molecules containing a late, interferon alpha-2a, interferon alpha-2b, irinotecan, nucleotide sequence identical to a portion of the target gene leflunomide, letrozole, leucovorin, levamisole, lomustine, are preferred for inhibition. RNA sequences with insertions, meclorethamine (nitrogen mustard), megestrol acetate, mel deletions, and single point mutations relative to the target phalan, mercaptopurine, mesna, methotrexate, methoXSalen, sequence have also been found to be effective for inhibition. mitomycin C, mitotane, mitoxantrone, mycophenolate Thus, sequence identity may optimized by sequence com mofetil, nandrolone phenpropionate, nilutamide, nofetumo parison and alignment algorithms known in the art (see Grib mab, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pega skov and Devereux, Sequence Analysis Primer, Stockton demase, pegaspargase, pegfilgrastim, pentostatin, pipobro Press, 1991, and references cited therein) and calculating the man, plicamycin, porfimer Sodium, procarbazine, quinacrine, percent difference between the nucleotide sequences by, for rasburicase rituximab, SargramoStim, streptozocin, tacroli example, the Smith-Waterman algorithm as implemented in US 2011/O 1892.87 A1 Aug. 4, 2011
the BESTFIT software program using default parameters 0207. In some embodiments, the matrices, such as poly (e.g., University of Wisconsin Genetic Computing Group). mer multilayers, are nanoscale to microscale in dimension. Greater than 90% sequence identity, or even 100% sequence Accordingly, in some embodiments, the matrices are from identity, between the inhibitory RNA and the portion of the about 1 nm to 1000 nm thick, from about 1 nm to 500 nm. target gene is preferred. Alternatively, the duplex region of the thick, from about 1 nm to 100 nm thick, from about 1 nm to RNA may be defined functionally as a nucleotide sequence about 25 nm thick, from about 1 nm to about 10 nm thick, or that is capable of hybridizing with a portion of the target gene less than about 500 nm, 100 nm, 25 nm or 10 nm thick. It is transcript. contemplated that the nanoscale dimension of the matrices 0204 There is no upper limit on the length of the dsRNA (i.e., the nanoscale thickness) allows for the loading of a that can be used. For example, the dsRNA can range from lower total amount of an active agent while still allowing about 21 base pairs (bp) of the gene to the full length of the delivery of an effective amount (i.e., an amount of active gene or more. In one embodiment, the dsRNA used in the agent that accelerates wound healing as compared to con methods of the present invention is about 1000 by in length. In trols) of the active agent as compared to matrix structures another embodiment, the dsRNA is about 500 by in length. In with greater thickness. It is contemplated that the lower total yet another embodiment, the dsRNA is about 22 by in length. loading levels result in reduced toxicity in the wound envi In some preferred embodiments, the sequences that mediate ronment, especially when antimicrobial compounds are RNAi are from about 21 to about 23 nucleotides. The isolated incorporated into the polymer multilayer. iRNAs of the present invention mediate degradation of the 0208. In some embodiments, the compliance of the matri target RNA. ces, such as polymer multilayers, is adjusted to facilitate cell 0205 The double stranded RNA of the present invention migration in the wound. In some embodiments, the matrices need only be sufficiently similar to natural RNA that it has the have a compliance, measured in kilopascals (kPa) of from ability to mediate RNAi for the target RNA. In one embodi about 3 to about 500 kPa, about 7 to about 250 kPa, about 10 ment, the present invention relates to RNA molecules of to about 250 kPA or from about 10 to about 200 kPa. In some varying lengths that direct cleavage of specific mRNA to embodiments, the matrices, e.g., polymer multilayers, have a which their sequence corresponds. It is not necessary that compliance of from about 3 kPa to 5, 4, 3, 2, 1, 0.5, 0.1 and there be perfect correspondence of the sequences, but the 0.05 GPa (gigapascals). correspondence must be sufficient to enable the RNA to direct (0209. A. Polymer Matrix Materials RNAi cleavage of the target mRNA. In a particular embodi 0210. In some embodiments, the matrix is a polymer mul ment, the RNA molecules of the present invention comprise a tilayer. In some embodiments, the multilayer structures com 3' hydroxyl group. prise layers of polyelectrolytes (i.e., forming a polyelectro lyte multilayer), while in other embodiments, the multilayers III. Matrix Compositions comprise polymers that do not have a charge (i.e., non-ionic 0206. In some embodiments, the present invention pro polymers) or a combination of charged and uncharged poly vides compositions comprising a matrix that can be applied to mer layers. In some embodiments, it is contemplated that a wound. In some embodiments, the matrix is functionalized. polyelectrolyte films built-up by the alternated adsorption of In some embodiments, the matrix comprises one or more cationic and anionic polyelectrolyte layers constitute a novel polymers, preferably biocompatible, or is formed from one and promising technique to modify wound Surfaces in a con more proteins, or is a combination of polymers and proteins. trolled way (Decher et al., 1992. Thin Solid Films 210/211: The matrix is preferably functionalized to allow for covalent 831; Decher, 1997, Science 277:1232). One of the most interaction and/or binding to the wound bed, or to allow important properties of such multilayers is that they exhibit an application of wound active agents to the matrix. In some excess of alternatively positive and negative charges (Caruso embodiments, a wound active agent, for example an antimi et al., 1999, JAm Chem Soc 121:6039; Ladam et al., 2000, crobial agent Such as a solver compound, is incorporated into Langmuir 16:1249). Not only can this constitute the motor of the matrix. In some embodiments, the matrix is formed or their buildup (Joanny, 1999, Eur. Phys.J. Biol. 9:117), but it provided on a solid Support. The Solid Support can form an allows, by simple contact, to adsorb a great variety of com outer permeable, semi-permeable, or impermeable barrier pounds Such as dyes, particles (Cassagneau et al., 1998, J. Am. after application of the matrix to a wound or can serve as a Chem. Soc. 120:7848; Caruso et al., 1999, Langmuir removed support for transfer and application of the matrix to 15:8276; Lvov et al., 1997, Langmuir 13:6195), clay micro the wound followed by removal of the support. The present plates (Ariga et al., 1999, Appl. Clay Sci. 15:137) and pro invention is not limited to any particular Solid Support. teins (Keller et al., 1994, J. Am. Chem. Soc. 116:8817: Lvov Indeed, a variety of Solid Supports are contemplated, includ et al., 1995, J. Am. Chem. Soc. 117:61 17: Caruso et al., 1997, ing, but not limited to, Solid Supports formed from elasto Langmuir 13:3427). meric materials such PDMS, nylon, Teflon R, Gortex R, silk, 0211 Polyelectrolyte layers are formed by alternating polyurethane, silicon, preferably medical grade silicon, applications of anionic polyelectrolytes and cationic poly PVDF, and solids supports formed from polyvinyl alcohol electrolytes to surfaces to form a polyelectrolyte multilayer. and polyethylene oxide. In some embodiments, the Solid Sup In some embodiments, one or more wound active agents, such port is a wound dressing that is compatible with functional as those described above, are incorporated into the multilayer. ization by addition of a matrix material. Examples of com Preferably, at least four layers, and, more preferably, at least mercially available wound dressings that can be modified by six layers are used to form the polyelectrolyte multilayer. addition of a matrix as described below include, but are not 0212 Cationic polyelectrolytes useful in the present limited to, BiobraneTM, gauze, adhesive tape, bandages such invention can be any biocompatible water-soluble polyca as Band-Aids(R), and other commercially available wound tionic polymer, for example, any polymer having protonated dressings including but not limited to COMPEEL(R), DUO heterocycles attached as pendant groups. As used herein, DERMTM, TAGADERMTM and OPSITE(R). “water soluble” means that the entire polymer must be soluble US 2011/O 1892.87 A1 Aug. 4, 2011 34 in aqueous solutions, such as buffered saline or buffered 0217. In some embodiments, the multilayer structures are saline with Small amounts of added organic solvents as co formed from uncharged polymers or from a combination of solvents, at a temperature between 20 and 37° Centigrade. In charged and uncharged polymers. Examples of uncharged some embodiments, the material will not be sufficiently polymers include, but are not limited to, dextran, dextran soluble (defined herein as soluble to the extent of at least one sulfate, diethylaminoethyl (DEAE)-dextran, hydroxyethyl gram per liter) in aqueous Solutions perse but can be brought cellulose, ethyl(hydroxyethyl) cellulose, acrylamide, poly into Solution by grafting the polycationic polymer with water ethylene oxide, polypropylene oxide, polyethylene oxide— soluble polynonionic materials such as polyethylene glycol. polypropylene oxide copolymers, PAAN, Ficoll, polyvi nylpyrolidine, and polyacrylic acid. 0213 Representative cationic polyelectrolytes include 0218. In some embodiments, the multilayer structures are natural and unnatural polyamino acids having net positive formed from one or more amphoteric polymers, alone in charge at neutral pH, positively charged polysaccharides, and combination with the other polymers described herein. In positively charged synthetic polymers. Examples of Suitable Some embodiments, the amphoteric polymers comprise one polycationic materials include polyamines having amine or more of acrylic acid (AA), DMAEMA (dimethylaminoet groups on either the polymer backbone or the polymer side hyl methacrylate). APA (2-aminopropyl acrylate), Mor chains, such as poly-L-lysine (PLL) and other positively phEMA (morpholinoethyl methacrylate), DEAEMA (diethy charged polyamino acids of natural or synthetic amino acids laminoethyl methacrylate), t-ButylAEMA or mixtures of amino acids, including, but not limited to, (t-butylaminoethyl methacrylate), PipEMA (piperidinoethyl poly(D-lysine), poly(ornithine), poly(arginine), and poly methacrylate), AEMA (aminoethyl methacrylate), HEMA (histidine), and nonpeptide polyamines such as poly(ami (2-hydroxyethyl methacrylate), MA (methyl acrylate), MAA nostyrene), poly(aminoacrylate), poly (N-methylaminoacry (methacrylic acid) APMA (2-aminopropyl methacrylate), late), poly (N-ethylaminoacrylate), poly(N,N-dimethyl AEA (aminoethyl acrylate). In some embodiments, the aminoacrylate), poly(N,N-diethylaminoacrylate), poly(ami amphoteric polymer comprises (a) carboxylic acid, (b) pri nomethacrylate), poly(N-methyl amino-methacrylate), poly mary amine, and (c) secondary and/or tertiary amine. The (N-ethyl aminomethacrylate), poly(N,N-dimethyl ami amphoteric polymers have an isoelectric point of 4 to 8, nomethacrylate), poly(N,N-diethyl aminomethacrylate), preferably 5 to 7 and have a number average molecular weight poly(ethyleneimine), polymers of quaternary amines, such as in the range of 10,000 to 150,000. poly(N.N.N-trimethylaminoacrylate chloride), poly(methy 0219 Polymer layers may beformed by a variety of meth acrylamidopropyltrimethylammonium chloride), and natural ods. In some embodiments, the polymer layers are formed on or synthetic polysaccharides such as chitosan. solid supports as described in detail below. In some embodi 0214. In general, the polymers must include at least five ments, it is contemplated that the polymer or polymer multi charges, and the molecular weight of the polycationic mate layers is formed by sequential application of polymers using rial must be sufficient to yield the desired degree of binding to either a pump (including Syringes, inkjet printers, and elec a tissue or other Surface, having a molecular weight of at least trojets) or spray, Such as an aerosol spray. In other embodi 1000 g/mole. ments, particle bombardment is utilized. In other embodi 0215 Polyanionic materials useful in the present inven ments, the use of a brush including an air brush is tion can be any biocompatible water-soluble polyanionic contemplated. In other embodiments, a sponge is utilized. In polymer, for example, any polymer having carboxylic acid other embodiments a Solid Support or stamp Such as an elas groups attached as pendant groups. Suitable materials include tomeric material, for example, PDMS (polydimethylsilox alginate, carrageenan, furcellaran, pectin, Xanthan, hyalu ane), silicone, hydrogel or latex, is used to Support the poly ronic acid, heparin, heparan Sulfate, chondroitin Sulfate, der merlayer and mechanically transfer the polymer layer into or matan Sulfate, dextran Sulfate, poly(meth)acrylic acid, oxi onto the wound bed. dized cellulose, carboxymethyl cellulose and 0220. In some embodiments, the matrix comprises one or crosmarmelose, synthetic polymers and copolymers contain more proteins. In preferred embodiments, the proteins form a ing pendant carboxyl groups, such as those containing maleic hydrogel. In some preferred embodiments, the matrix com acid or fumaric acid in the backbone. Polyaminoacids of prises one or more extracellular matrix proteins. In some predominantly negative charge are also Suitable. Examples of embodiments, the matrix comprises at least one of collagen, these materials include polyaspartic acid, polyglutamic acid, laminin, vitronectin, fibronectin, keratin, and combinations and copolymers thereof with other natural and unnatural thereof. As described above, the protein matrix may prefer amino acids. Polyphenolic materials such as tannins and ably be formed by a variety of methods. In some embodi lignins can be used if they are sufficiently biocompatible. ments, the protein matrix is formed on Solid Supports as Preferred materials include alginate, pectin, carboxymethyl described in detail below. In some embodiments, it is con cellulose, heparin and hyaluronic acid. templated that the protein matrix is formed by application of 0216. In some embodiments, the cationic polyelectrolyte proteins or Solutions or gels of proteins using either a pump used is PLL and the anionic polyelectrolyte used is poly(L- (including syringes, inkjet printers, and electrojets) or spray, glutamic acid) (PGA). Indeed, the use of a variety of poly Such as an aerosol spray. In other embodiments, the use of a electrolytes is contemplated, including, but not limited to, brush including an air brush is contemplated. In other poly(ethylene imine) (PEI), poly(allylamine hydrochloride) embodiments, a sponge is utilized. In other embodiments a (PAH), poly(sodium 4-styrenesulfonate) (PSS), poly(acrylic Solid Support or stamp Such as an elastomeric material, for acid) (PAC), poly(maleic acid-co-propylene) (PMA-P), and example, PDMS (polydimethylsiloxane), silicone, hydrogel poly(vinyl sulfate) (PVS). It is also possible to use naturally or latex, is used to Support the protein matrix and mechani occurring polyelectrolytes, including hyaluronic acid and cally transfer the protein matrix into or onto the wound bed. chondroitin sulfate. In still further embodiments, the polymer 0221. In some embodiments, the matrix is further modi is a dendrimer, grafted polymer, or star architecture polymer. fied to include cells, including, but not limited to, stem cells, US 2011/O 1892.87 A1 Aug. 4, 2011
keratinocytes, 3-D skin constructs, corneal epithelial cells, 800 nm in diameter. In some embodiments, beads and other conjunctival cells, corneal limbal stem cell, human embry spherical and non-spherical particles contain their own Sur onic stem cells, pluripotential stem cells, adult induced stem face topography. For example, the bead surface may comprise cells, hematopoietic stem cells, hepatocytes, pancreatic cells undulations, indentations, tunnels, holes, knobs, ridges, etc. and the like. In some embodiments, the compositions and Spherical particles, such as beads, may or may not be inert methods described herein are used to functionalize harvested and may, for example, be magnetic. Magnetic beads com and processed (frozen, freeze dried, fixed and stored dry or prise, for example, a magnetic core or magnetic particles. wet, fresh tissue for direct transplant) animal and human Examples of different bead compositions are found at, for tissue (pig?human aortic valve, harvested human amniotic example, U.S. Pat. Nos. 5.268,178, 6.458,858, 6.869,858, membrane, human cadaver skin, harvested Sclera and cornea, and 5,834,121, each of which is incorporated herein by ref harvested cadaver bone, harvested blood vessels and the like. erence in its entirety. In some embodiments, the particles In some embodiments, the compositions and methods comprise or are impregnated with a wound active agent. described herein are used to functionalize skin constructs, 0226. It is contemplated that polymer multilayers such as including, but not limited to, autograft skin (i e , skin is those described above offer the opportunity to chemically and harvested from a patient, functionalized as described herein, topographically engineer the Surface of Substrates. Using and returned to the patient), organotypically cultured human existing technologies, one has to construct nanostructures or skin equivalents, and other keratinocyte products such as organic films (like polymer-multilayers) onto those Surfaces, DermagraftTM. and dope them with active agents. Since these procedures are 0222 B. Polymer Multilayers Associated with Microscale laborious (it can take 6 hrs to fabricate a multilayer films or Nanoscale Beads containing 20 layers, which is prohibitively long for say treat 0223) In some embodiments, the present invention pro ment of a wound), can involve harsh chemicals, Solutions vides polymer multilayers that comprise nano- or microscale having non-physiological pHs, and possible mechanical particles. In some embodiments, the particles are dispersed in shear forces, they may damage or compromise the Substrates the polymer multilayer, for example, in between the polymer on which they are formed (particularly biological tissues and layers. In other embodiments, the particles are displayed on a sensitive biomedical devices). To overcome these limitations, Surface. Such as a top or a bottom surface, of the polymer in some embodiments, the polymer multilayers of the present multilayer. In some embodiments, the particles are associated invention are pre-fabricated on elastomeric stamps (such as with said polymer multilayer in a manner selected from the PDMS stamps) and then mechanically transferred to a sub group consisting of interspersed or dispersed in said polymer strate of interest by stamping onto the substrates of interest. multilayer, displayed on a surface of said polymer multilayer, Although the concept of contact printing pre-fabricated nano and underneath the polymer multilayer. and microstructures on hard Substrates (microscope slides, 0224. In some embodiments, the polymer multilayers are silicon wafers) has been employed for microfabrication of formed as described above. In some preferred embodiments, electromechanical systems; those methods fail when used on the particles have diameter or other linear measurement of a Soft Substrates, particularly Substrates with mechanical prop feature, such as a length or width, which is greater than the erties typically of a range of biological tissues. thickness of polymer multilayer. In some embodiments, the 0227. The present invention provides methods and com diameter or other linear measurement is from about 1, 1.5, 2, positions of matter that make possible the transfer of organic 3, or 4 times greater than the thickness of the polymer multi and inorganic thin films, including polymer multilayers, onto layer up to about 5, 10 or 20 times greater than thickness of the compliant Substrates, including soft-substrates with mechani polymer multilayer. cal properties equivalent to those of biological tissues (elastic 0225. In some embodiments, nanometer to micrometer modulus, E, of about 1-500 kPa preferably about 10-100 sized biocompatible particles, such as spherical (e.g., beads) kPa). Existing methods described in literature which allow and/or non-spherical (e.g., oblongs, needles, cubes, tetrahe transfer of organic thin films onto rigid substrates like glass dral, mushroom-like structures, haybale-like structures) par (E-65 GPa) with various surface chemistries, fail when used ticles or electrospun polymers, are utilized in or on the poly on soft-surfaces (E-100 kPa). FIG. 11 demonstrates these mer multilayer. Microbeads have a size generally ranging observations. The method demonstrated in Example 14 and from about 1 to about 500 micrometers, while nanobeads FIG. 12 facilitates transfer of polymer-multilayers onto soft have a size generally ranging from about 1 to about 1000 Surfaces, including biological tissues, including skin-grafts nanometers. Microbeads may further comprise micro- (i.e., and mouse pelts. Incorporation of polymer-microspheres from 1 to 100 micrometers) or nano-scale (0.1 to 1000 (E-3 GPa) within an assembly of polymer-multilayers on an nanometer) features on the surface of the bead. Nanobeads elastomeric stamp, when stamped onto a human skin-graft or may further comprise nanoscale features (i.e., 0.1 to 500 a biomedical grade soft silicone elastomer (E-100 kPa), nanometer features) on the surface of the bead. In some leads to a substantially complete transfer of rigid-micro embodiments, a wound active agent is present in the form of spheres and polymer-multilayers in contact with the micro a bead or particle. Such as silver nanoparticles. In some spheres. embodiments, the biocompatible particles are biodegradable. 0228. This method allows engineering the surface of soft In some embodiments, the biocompatible particles, for substrates (E-100 kPa) by contact printing pre-fabricated example, are modified with Surface chemistry cross-linkers functionalized polymer-multilayers onto their surface. This that allow attachment and immobilization of wound active opens up a vast area of opportunities to chemically and topo agents, extracellular matrix compounds, etc. as described graphically modify surfaces of biological tissues and soft elsewhere herein. In some embodiments, the size of the bio implantable biomedical devices, like biomedical grade sili compatible particles. Such as beads, are at least lnm, at least 5 cone, and generate surfaces with tailored functionality, chem nm, at least 10 nm, at least 20 nm, at least 50 nm, at least 100 istry and topography at nanometer Scale. This is a versatile nm, at least 200 nm, at least 300 nm, at least500 nm, or at least Solution to engineer or pattern, for example, a wound-bed, US 2011/O 1892.87 A1 Aug. 4, 2011 36 from two perspectives. First, from a product development with active agents. Such immobilization of active agents in view-point, fabrication and functionalization of polymer polymer-multilayers over wound-bed permits: 1) Precise multilayers used in this approach can be easily automated in delivery and minimal loading of active molecules onto the a manufacturing environment using robots. Second, from wound-bed, thereby providing efficacious wound treatment application view-point, an end-user would simply have to without cytotoxic effects and at reduced costs. 2) Prolong contact-print materials from the stamps onto the Substrates, bioavailability of the active agents in the wound-bed, circum instead of preparing multilayers on the Substrates. venting frequent dressings and thus reducing patient pain and 0229. Accordingly, the present invention provides meth overall costs of wound-management. This is particularly sig ods to tailor the Surface chemistry and nanostructured topog nificant in the treatment of chronic wounds that take several raphy of soft-substrates by mechanical transfer of pre-fabri months to heal. 3) Controlled delivery of bioactive molecules, cated, nanometer-thick organic films, impregnated with both spatially and temporally. This allows regulating the dis active agents, onto the Substrates. This technology provides tribution of active molecules across the wound-bed and cre unprecedented advantages over several competing existing ating their gradients. For example, invitro studies have shown technologies. First, technology described in past literature for that gradients of growth-factors promote the migration of stamping pre-fabricated polymer-multilayer films onto Sub cells involved in wound-healing to the wound-bed and can strates relevant for the microfabrication of electromechanical expedite wound-closure. 4) Putting active molecules directly systems does not allow their transfer onto soft-substrates with into the cellular microenvironment, increasing their local mechanical strength relevant to biological tissues. Thus, the concentration and avoiding problems of mass transfer limi present invention opens up the possibilities of engineering tations or complex aggregation encountered in release sys surfaces of a whole new vast arena of soft substrates by tems. 5) Maintaining the active molecules locally in the contact-printing pre-fabricated functionalized polymer matrix, limiting any systemic loss or potential undesirable films. Some of such soft-substrates relevant for immediate diffusion to distant sites. commercialization of this technology include biological tis 0233. This approach to wound-healing using current Sues, wound-beds, and implantable biomedical devices like invention is fundamentally new in the sense that it attempts to biomedical grade silicone. Furthermore, the study performed engineer the wound-bed. The wound-bed surface is modi during this invention leads to the new observation that fied chemically and topographically using nanometer-thick mechanical properties of the substrates influence the organic films impregnated with minimal concentrations of mechanical transfer of organic films onto them- these new active agents. This contrasts to currently practiced principles of mechanical transfer can be used to selective approaches which attempt to treat the wound by drizzling transfer organic films on Substrates with, for example, pat high concentrations of bioactive agents over the wound. terned soft and hard regions. 0234. Accordingly, in some embodiments, the present 0230 Second, to chemically or topographically modify invention provides a polymer multilayer comprising at least the surface of soft-substrates (E-100 kPa) using existing one polymer, the polymer multilayer having associated there technologies, one has to construct nanostructures or organic with particles selected from the group consisting of nanoscale films (like polymer-multilayers) onto those Surfaces, and and microscale particles. In some embodiments, the polymer dope them with active agents. Since these procedures mostly multilayer is from 1 nm to 1000 nm thick and in preferred involve harsh chemicals, Solutions at non-physiological pHs, embodiments, from about 1 to about 250 nm thick. In some and possible mechanical shear forces, they may disrupt the embodiments, the polymer multilayer has a compliance of sensitive-substrates, particularly biological tissues and sensi from 3 to 500 kPa. In some embodiments, the polymer mul tive biomedical devices. The invention described here allows tilayer is disposed on a Support. In some embodiments, the to contact-print pre-fabricated polymer-multilayers impreg polymer multilayer is formed on a Support. In some preferred nated with active agents onto the Soft-substrates, instead of embodiments, the Support is an elastomeric Support. assembling them on those substrates. Furthermore, the direct 0235. In some embodiments, the present invention pro stamping of pre-fabricated polymer-multilayers onto the Sub vides methods for modifying a Surface comprising contacting strates provide precise control over the two and three-dimen a surface with a polymer multilayer as described above under sional placement of the polymer-multilayers. By sequential conditions such that said polymer multilayer is transferred to stamping of multiple, pre-fabricated polymer-multilayers said Surface. In some embodiments, the Surface is a soft using the methodology, multilevel and multi-component pat Surface. In some embodiments, the Surface has an elastic tern structures can be conveniently introduced onto device modulus of from about 1 to about 500 kPa, preferably about Surfaces. Thus, this invention provides unprecedented control 10 to about 100 kPa. In some embodiments, the surface is a at nanometers-level over the surface-modification of soft biological Surface. In some embodiments, the biological Sur Substrates. face comprises proteins, lipids and/or carbohydrates. In some 0231. Third, in the context of wound-healing, current embodiments, the biological surface is selected from the clinical wound-management approaches involve delivering group consisting of skin, a wound bed, hair, a tissue surface, active agents to the wounds in ointments or dressings. The and an organ Surface. In some embodiments, the Surface is a wound-dressings are reservoirs of active agents and require biologically compatible surface, or the surface of a biomedi high loading concentrations for the active agents to diffuse cal device. In some embodiments, the Surface is a silicone through the wound-fluid and reach the wound-bed where Surface. activity is required. High concentrations of active agents are 0236 C. Functionalization Agents cytotoxic, causing tissue toxicity and impaired wound-heal 0237. In some embodiments, the matrices described above ing. are functionalized. In preferred embodiments, the matrices 0232. The present invention allows immobilizing active are functionalized with one or more covalent modification agents onto the Surface of the wound-beds by stamping pre agents. In some preferred embodiments, the crosslinkers fabricated nanostructured polymer-multilayers impregnated comprise either an azide group or an alkyne group so that US 2011/O 1892.87 A1 Aug. 4, 2011 37 suitable click chemistries can be utilized. In some embodi 0239. The covalent modification agent may be applied to ments, the at least one covalent modification agent is a homo the matrix by any suitable method. In some embodiments, it bifunctional cross-linker. In other embodiments, the at least is contemplated that the covalent modification agent is one covalent modification agent is a heterobifunctional cross applied using either a pump (including Syringes, inkjet print linker. For example, in some embodiments, the homobifunc ers, and electrojets) or spray, Such as an aerosol spray. In other tional cross-linker is an N-hydroxySuccinimidyl ester (e.g., embodiments, the use of a brush including an air brush is including, but not limited to, disuccinimidyl ester, dithiobis contemplated. In other embodiments, a sponge is utilized. (succinimidylpropionate), 3,3'-dithiobis(sulfosuccinimidyl 0240 Following application of the covalent modification propionate), disuccinimidyl Suberate, bis(SulfoSuccinimidyl) agent to the matrix, a functionalized matrix is formed. In Suberate, disuccinimidyl tartarate, disulfo Succinimidyl Some embodiments, the functionalized matrix displays one or tartarate, bis 2-(Succinimidyloxycarbonyloxy)ethylsulfone, more reactive moieties, for example, azide moieties, succin bis(2-(sulfosuccinimidooxycarbonyloxy)ethylsulfone, eth imidyl moieties, alkyne moieties or any of the other reactive ylene glycolbis(Succinimidylsuccinate), ethylene glycolbis groups of the compounds described above. (SulfoSuccinimidylsuccinate), disuccinimidyl glutarate, and 0241. In some embodiments, the matrix is modified by N,N'-disuccinimidylcarbonate). In some embodiments, the PEGylation with polyethylene glycol. It is contemplated that homobifunctional cross-linker is at a concentration between 1 PEGylation can be used to control interaction of the matrix nanomolar and 10 millimolar. In some preferred embodi with the wound bed and to prevent nonspecific adsorption as ments, the homobifunctional cross-linker is at a concentra desired. PEGylation can also be used to control delivery of the tion between 10 micromolar and 1 millimolar. In other wound active agent from the matrix. embodiments, the at least one covalent modification agent is 0242 D. Wound Active Agents a heterobifunctional cross-linker (e.g., including, but not lim 0243 In some embodiments, the matrices comprise one or ited to, N-Succinimidyl 3-(2-pyridyldithio)propionate. Suc more wound active agents as described in detail above. In cinimidyl 6-(3-2-pyridyldithio-propionamido)hexanoate, Some embodiments, the wound active agent or agents are sulfosuccinimidyl 6-(3'-2-pyridyldithio-propionamido) noncovalently incorporated into the matrix. In some preferred hexanoate, Succinimidyloxycarbonyl-O-methyl-O-(2-py embodiments, silver containing antimicrobials are incorpo ridyldithio)toluene, sulfosuccinimidyl-6-O-methyl-O-(2- rated into the functionalized matrix, for example, a polyelec pyridyldithio)toluamidohexanoate, succinimidyl 4-(N- trolyte multilayer as described above. In some embodiments, maleimidomethyl)cyclohexane-1-carboxylate, the wound active agent or agents are covalently immobilized Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1- on the matrix, for example, via a covalent modification agent. carboxylate, m-maleimidobenzoyl-N-hydroxySuccinimide 0244. In some embodiments, the one or more wound ester, m-maleimidobenzoyl-N-hydroxy-sulfosuccinimide active agents are applied to form a gradient with respect to the ester, N-Succinimidyl(4-iodoacetyl)aminobenzoate, Sulfo wound modifying agent. In general, the gradients present a Succinimidyl(4-iodoacetyl)aminobenzoate, Succinimidyl-4- higher contraction of wound active agent at one or more first (p-maleimidophenyl)butyrate, SulfoSuccinimidyl-4-(p-male desired locations in the wound following application of the imidophenyl)butyrate, N-(y-maleimidobutyryloxy) wound modifying agent to the wound and a lower concentra Succinimide ester, N-(y-maleimidobutyryloxy) tion of wound active agent at one or second location in the SulfoSuccinimide ester, Succinimidyl 6-((iodoacetyl)amino) wound following application of the matrix to the wound. For hexanoate, Succinimidyl 6-(6-((((4-iodoacetyl)amino) example, the concentrations of the wound active agents are hexanoyl)amino)hexanoate. Succinimidyl 4-(((iodoacetyl) layered in a wound bed in a gradient Such that higher concen amino)methyl)cyclohexane-1-carboxylate, Succinimidyl trations of a particular composition is greater proximal to the 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl) wound bed than distal to the wound bed in a vertical fashion. amino)-hexanoate, and p-nitrophenyl iodoacetate). In some The converse, where concentrations of compositions is embodiments, the heterobifunctional cross-linker is modified greater distal to the woundbed than proximal, is also contem with functional groups, rendering it soluble in aqueous sol plated. Concentration of compositions in a wound bed vents for delivery as an aqueous solution. Furthermore, in wherein a horizontal gradient is deposited is also contem Some embodiments, the aqueous solution contains additives plated. Topographical gradients are also contemplated, (e.g., including, but not limited to, Surfactants and block wherein compositions are deposited Such that the concentra copolymers). In other embodiments, a multiplicity of hetero tions of compositions in a wound bed or on a biocompatible bifunctional cross-linkers can be attached to a molecule, particle follow the topography of the Substrate, for example, a polymer or particle to serve as the cross-linking agent. In higher concentration of compositions is deposited in the val other embodiments, the heterobifunctional cross-linker is leys of undulations of an exemplary Substrate compared to the dissolved in an organic solvent (e.g., including, but not lim peaks of the undulations. ited to, dimethyl sulfoxide). 0245. In some embodiments, the gradient comprises a 0238. In some embodiments, the covalent modifier is a higher concentration of the wound active agent in the center photoactivatable crosslinker. Suitable photoactivatable of the matrix which transitions to a lower concentration of the crosslinkers include, but are not limited to, aryl azide N-((2- wound active agent away from the center of the matrix. pyridyldithio)ethyl)-4-azidosalicylamide, 4-azido-2,3,5,6- Accordingly, when the matrix is applied to a wound, the tetrafluorobenzoic acid, Succinimidyl ester, 4-azido-2,3,5,6- gradient results in a higher concentration of wound active tetrafluorobenzoic acid, STP ester, benzophenone agent in the center of the wound and a lower concentration of maleimide, Succinimidyl ester of 4-benzoylbenzoic acid, wound active agent as one moves to the periphery of the N-5-Azido-2-Nitrobenzoyloxysuccinimide, N-Hydroxysul wound. In some embodiments, the gradient comprises a lower fosuccinimidyl-4-azidobenzoate, N-Hydroxysuccinimidyl concentration of the wound active agent in the center of the 4-azidosalicylic acid, and (4-p-AZidosalicylamidobuty matrix which transitions to a higher concentration of the lamine). wound active agent away from the center of the matrix. US 2011/O 1892.87 A1 Aug. 4, 2011
Accordingly, the gradient results in a lower concentration of embodiments, the matrices preferably comprise one more wound active agent in the center of the wound and a higher wound active agents as described above and/or or nanoscale concentration of wound active agent as one moves to the or microscale beads as described above. In some preferred periphery of the wound. If two or more wound active agents embodiments, the wound active agent is one or more of silver are utilized, they can be presented as similar gradients or the nanoparticles, elemental silver, and silver containing com gradients can be varied so that the concentrations of the two or pounds such as silver Sulfadiazine and related compounds, more wound active agents vary across the wound. The gradi preferably included in the concentration ranges described ents of high or low concentration can be any shape, such as above. circular, square, rectangular, oval, oblong, etc. So that the 0249. In some embodiments, the present invention pro matrix and gradient can conform to a variety of wound vides methods for transferring a polymer multilayer to a shapes. For example, for long, incision type wound, the gra desired surface, Such as soft Surface. Such soft Surfaces dient may be centered on a longitudinal axis that extends include, but are not limited to, skin, a wound bed, a tissue, along the length of the wound and can be centered on the artificial tissues including artificial skin tissues such as orga wound. As another example, the gradient can be circular or notypically cultured skin tissues, Apligraf R, Dermagraft(R), oval-shaped for application to open type wounds, burns, Sores Oasis.R., Transcyte R, Cryoskin R) and Myskin R, artificial tis and ulcers that are roughly circular or oval. In other embodi Sue matrices, gels comprising biomolecules, a wound dress ments, the gradients comprise a series of features arranged in ing, and a biologic wound dressing. In some embodiments, a pattern. For example, the gradients can form a series of the desired surface is contacted with a polymer multilayer, stripes or high and low concentrations of one or more wound e.g., a polymer multilayer Supported on a Support and pres active agents along a longitudinal axis of the matrix. Alterna sure is applied to effect transfer of the polymer multilayer tively, the gradients can form a checkerboard pattern, array, from the support to the desired surface. In some embodi concentric circles, overlapping circles or oval, etc. ments, the pressure is from about 10 to about 500 kPa. In some 0246 E. Support Materials embodiments, the transfer is performed in the substantial, or 0247. In some embodiments, the matrices are formed on a complete, absence of solution. Such dry transfer processes do Support material. Suitable Support materials include, but not not involve exposure of biological components of the desired limited to, Solid Supports formed from polymeric materials, Surface to aqueous Solutions containing species that may elastomeric materials such PDMS, nylon, Teflon R, Gortex R, influence the activity of the biological components. In some silk, polyurethane, and silicon, preferably medical grade sili embodiments, the transfer is performed through a gas phase. con, PVDF, polyethylene oxide, and water soluble materials In some embodiments, the transfer is performed in an envi and polymers such as polyvinyl alcohol. In some embodi ronment where the humidity is less than 100% of saturation. ments, the polymer(s) used to form the polymer multilayer In some embodiments, the transfer is performed in the are different than the polymer(s) forming the Support. In some absence of liquid water. embodiments, an additional layer of material. Such as a water 0250. Accordingly, in some embodiments, the present soluble material or polymer, is disposed between the polymer invention provides wound dressings comprising a Support multilayer and the Support. In some embodiments, the Solid material having a surface oriented to a wound, wherein the Support is a wound dressing or biologic wound dressing that surface oriented to the wound is modified with a matrix mate is compatible with functionalization by addition of a matrix rial of the present invention. When applied to a wound, the material. Examples of commercially available wound dress surface of the support material modified with the matrix mate ings that can be modified by addition of a matrix as described rial is put into contact with the wound bed. below include, but are not limited to, BiobraneTM, gauze, 0251. In some embodiments, the support is a biologic adhesive tape, bandages such as BandAids(R), and other com wound dressing. In some embodiments, the biologic wound mercially available wound dressings including but not limited dressing is a type of wound dressing that comprises, e.g., is to COMPEEL(R), DUODERMTM, TAGADERMTM and coated with or incorporates, cells (e.g., keratinocytes or fibro OPSITE(R). In some embodiments, the support is a silicon blasts and combinations thereof) and/or one or more biomol wafer, Surfaces, such as glass or silicon wafers, that are coated ecules or fragments of biomolecules that can be placed in with hydrophobic self assembled monolayer, fabrics, such as contact with the wound surface. The biomolecules may be nylon fabrics, absorbent material, foam, a transparent thin provided in the form of an artificial tissue matrix omprising film, a hydrogel, hydrofibers, hydrocolloids, alginate fibers, one or more biomolecules. Examples of such biomolecules silica gel, Sodium polyacrylate, potassium polyacrylamides include, but are not limited, to collagen, glycosaminoglycans, and combinations thereof. In some embodiments, the Sup hyaluronic acid, laminin, vitronectin, fibronectin, keratin, ports comprise (e.g., is coated with or displayS) a biological antimicrobial polypeptides and combinations thereof. molecule selected from the group consisting of collagen, Examples of Suitable biologic wound dressings include, but hyaluronic acid, glycosaminoglycans, keratin, fibronectin, are not limited to, BIOBRANETM, IntegraTM, Apligraf R, Der vitronectin, laminin, and combinations or fragments thereof. magraft(R), Oasis.(R), Transcyte?R), Cryoskin R and Myskin R. 0248. The matrices described above can be synthesized 0252. In some embodiments, the support material is a directly on the support material or transferred to the support biosynthetic wound dressing constructed of an elastomeric material following synthesis, for example by transfer or film (e.g., a silicone film) Supported on Support material. Such stamping from a Suitable Support, such as an elastomeric as a fabric, preferably a polymeric fabric Such as a nylon support. There are several different methods that can be used fabric. In some embodiments, the fabric is at least partially to deposit polymer multilayers on a Support. In some embodi imbedded into the film (e.g., BioBraineTM). In some embodi ments, the polymer multilayers are deposited onto a Support ments, the elastomeric film is coated with one or more bio by adsorption from solution, spraying, or spin coating. As materials, for example collagen, keratin, fibronectin, Vit described above, the matrices are preferably polymer multi ronectin, laminin and combinations thereof. Accordingly, the layers, for example, polyelectrolyte multilayers. In some fabric presents to the wound bed a complex 3-D structure to US 2011/O 1892.87 A1 Aug. 4, 2011 39 which a biomaterial (e.g., collagen) has been bound, prefer stamp material, such as PDMS, and then transferred to the ably chemically bound. In some preferred embodiments, the Solid Support by a stamping process as described in detail surface presented to the wound is further modified with a above. matrix material as described above. In some preferred 0255 F. Use of Matrices embodiments, the matrix material is a polyelectrolyte mem 0256 In some embodiments, a matrix as described above brane comprising a wound active agent, preferably selected is applied to a wound under conditions such that wound from one or more of silver nanoparticles, elemental silver, and healing, as measured by wound contraction, is accelerated. In silver containing compounds such as silver Sulfadiazine and Some embodiments, the matrix is functionalized just prior to related compounds, and preferably included in the concen application to a wound, while in other embodiments, the matrix is provided in a sterilepackage and is functionalized so tration ranges described above. In some embodiments, the that it is ready for application following removal from the matrix further comprises nanoscale or microscale particles. sterile packaging. In some embodiments, the wound is pre The matrix can be synthesized directly on the collagen modi treated or primed with a covalent modification agent that is fied surface or transferred to the collagen modified surface by reactive with the functionalization agent displayed by the stamping as described above. functionalized matrix. For example, the wound may be modi 0253) In some embodiments, the support material is an fied as described in detail above with a covalent modification adhesive bandage comprising an adhesive portion (such as an agent displaying a reactive azide group, while the matrix may adhesive strip) and an absorbent material, preferably treated be modified with a covalent modification agent displaying an or coated with a material (i.e., a non-adherent material) to alkyne group. In general, the wound may be modified with prevent adhesion to the wound or comprising a layer of non covalent modification agent A which displays reactive group adherent material, such as Teflon R, on the surface of the X and the matrix displays covalent modification agent B absorbent pad that will contact the wound. In some embodi which displays reactive group Y, wherein X and Y react with ments, the Support material is an absorbent pad (e.g., a gauze each other to form a covalent bond. pad or polymer foam) preferably treated or coated with a 0257. In some embodiments, the matrices are provided as material (i.e., a non-adherent material) to prevent adhesion to kits, preferably with the matrix in a sterile package. In some the wound or comprising a layer of non-adherent material, embodiments, the matrix in the kit is pre-functionalized, such as Teflon R or other suitable material, on the surface of while in other embodiments, the matrix is not functionalized the absorbent pad that will contact the wound. In some and the kit comprises at least one functionalization agent embodiments, the non-adhesive material or layer is breath along with instructions on how to functionalize the matrix. In able. In some embodiments, the wound dressing comprises a Some embodiments, the kits comprise a functionalization gel-forming agent, for example, a hydrocolloid such as agent for functionalizing the wound bed prior to application Sodium carboxymethylcellulose. In some embodiments, the of the matrix. In some embodiments the matrix providing in absorbent pads or gel-forming agents are affixed to a material the kit comprises at least one wound active agent. In other that is waterproof and/or breathable. Examples include, but embodiments, the kits comprise a wound active agent and are not limited, semipermeable polyurethane films. The instructions from applying the wound active agent to the waterproof and/or breathable material may further comprise matrix prior to application to a wound. an adhesive material for securing the bandage to the skin of a subject. The waterproof and/or breathable material preferably IV. Methods of Treating Wounds forms the outer Surface of the adhesive bandage or pad, i.e., is 0258. A wound modifying agent with one or more wound the Surface opposite of the Surface comprising the matrix active agents or matrix, as described above can be applied to which contacts the wound. Examples of such adhesive ban all types of wounds. Furthermore, the compositions of the dages and absorbent pads include, but are not limited to, to present invention can be applied to all types of wounds. adhesive bandages and pads from the Band-AidR line of Furthermore, a wound modifying agent with one or more wound dressings, adhesive bandages and pads from the Nex wound active agents can be applied to skin, mucous mem care line of wound dressings, adhesive bandages and non branes, body cavities, and to internal Surfaces of bones, tis adhesive pads from the Kendall Curity Tefla?R line of wound Sues, etc. that have been damaged. A wound modifying agent dressings, adhesive bandages and pads from the TegadermR) with one or more wound active agents can be used on wounds line of wound dressings, adhesive bandages and pads from the Such as cuts, abrasions, ulcers, Surgical incision sites, burns, Steri-Strip(R) line of wound dressings, the COMFEEL(R) line and to treat other types of tissue damage. In some embodi of wound dressings, adhesive bandages and pads, the Duo ments of the present invention, the compositions and methods dermR line of wound dressings, adhesive bandages and pads, described above enhance wound healing. The present inven the TEGADERMTM line of wound dressings, adhesive ban tion contemplates that wound healing may be enhanced in a dages and pads, the OPSITE(R) line of wound dressings, adhe variety of ways. In some embodiments, the compositions and sive bandages and pads, adhesive bandages and pads from the methods minimize contracture of the wound as to best favor Allevyn TM line of wound dressings, adhesive bandages and function and cosmesis. In some embodiments, compositions pads from the Duoderm R) line of wound dressings, and adhe and methods promote wound contracture to best favor func sive bandages and pads from the Xeroform(R) line of wound tion and cosmesis. In some embodiments, the compositions dressings. and methods promote vascularization. In some embodiments, 0254. In some embodiments, the present invention pro the compositions and methods inhibit vascularization. In vides methods and systems for producing the articles Some embodiments, the compositions and methods promote described above. In some embodiments, the polyelectrolyte fibrosis. In some embodiments, the compositions and meth multilayers are directly deposited on the Solid Support by any ods inhibit fibrosis. In some embodiments, the compositions of the methods described in detail above. In some embodi and methods promote epithelial coverage. In some embodi ments, the polyelectrolyte multilayers are deposited onto a ments, the compositions and methods inhibit epithelial cov US 2011/O 1892.87 A1 Aug. 4, 2011 40 erage. In some embodiments, the compositions and methods acids, amines, and thiol groups found in the wound bed. of the present invention modulates one or properties of cells in Further, upon determination of optimal cross-linkers, the the wound environment or in the immediate vicinity of the immobilization of cytoactive agents and extracellular matrix wound. The properties that are modulated, e.g., are increased compounds as described herein are evaluated (e.g., using or decreased, include, but are not limited to adhesion, migra fluorescently labeled antibodies to immobilized compounds). tion, proliferation, differentiation, extracellular matrix secre It is contemplated that key parameters to a Successful ex vivo tion, phagocytosis, MMP activity, contraction, and combina evaluation include, but are not limited to, concentration of all tions thereof. immobilization compositions, buffer systems, and incubation times. Ex vivo evaluations constitute additional validation of 0259. The compositions of the present invention can be in vivo wound bed healing systems. covered with a secondary dressing, or bandage, if desired to 0263. In one embodiment, in vivo experiments further protect the layer or to provide additional moisture absorption, validate the wound bed healing methods as described herein. for example. In some embodiments, the diabetic transgenic mouse db/db 0260. If desirable, the wound modifying agent with one or are used, as a phenotype of the diabetic mice is impaired more wound active agents can be reapplied at a later time. It wound healing. Polyelectrolytes, cytoactive agents, and may be desirable to wound modifying agents having different extracellular matrices as described herein are applied to the formulations of wound active agents at different stages of db/db mice for optimization of wound healing. The present wound healing. invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice 0261. In some embodiments, the immobilization of cyto the present invention. Nonetheless, it is contemplated that active factors and matrices immobilized on a wound bed optimized wound healing in diabetic mice provides a model using, for example, polyelectrolytes are evaluated in vitro and system that correlates to wound healing for normal, cutane in vivo. In some embodiments, in vitro analysis comprising ous wounds. In one example, skin wounds are created in studying the ability of cytoactive factors to proliferate cell tandem on diabetic mice using a Surgical skin punch. After growth is evaluated in corneal epithelial cells and human wounding, test compounds are immobilized in one of the vascular endothelial cells. In some embodiments, in vitro wounds, leaving the other as a control (e.g., BSA only). The analysis comprises the construction of synthetic Surfaces that test compounds are evaluated, for example as described in mimic a wound bed, wherein said surfaces are created to Example 3, thereby providing further information for useful comprise Surface chemistries such as primary amine groups, in vivo compositions for wound bed healing. Information on carboxylic acids and thiol groups that are representative of the the impact of the test compounds on wound healing is also diversity of chemical functionality present in a wound bed. obtained by, for example, evaluation of the wound bed for For example, ECM constituents and cytoactive factors are epithelial coverage, extent of formation of granulation tissue, applied to the synthetic Surfaces, and immobilization is con collagen content, inflammation, fibroblast population and firmed by, for example, fluorescently labeled antibodies to the vascularization. Test compounds include, but are not limited ECMs and cytoactive factors thereby immobilized. Further in to, the polyelectrolytes as described herein, extracellular vitro analysis includes, but is not limited to, proliferation dose matrix components (e.g., collagen, laminin, MATRIGEL. response of the cytoactive factors and EMCs on the synthetic fibronectin, etc.) and cytoactive agents (e.g., EGF, VEGF, Surfaces. In some embodiments, the in vitro assays are used to PDGF). To further characterize the wound bed, confocal evaluate cytoactive factors that inhibit specific cell growth in microscopy is used to visualize cellular components in a a wound bed, thereby allowing for preferential recruitment three-dimensional space, thereby allowing for the visualiza and growth of those cell types optimal for wound healing and tion of the wound bed treatments in a native state. rapid return of tissue function and non-recruitment of antago 0264. In some embodiments, the present invention pro nistic cell species. vides for the development of personalized therapeutic proto 0262. In one embodiment, assays to evaluate immobiliza cols for wound healing. For example, the compositions as tion strategies are performed ex vivo. In some embodiments, described herein for wound healing are adapted for each cutaneous wounds are harvested from mice. For example, a 6 individual wound, taking into account, for example, environ mm Surgical skin punch is used to create a cutaneous defect mental factors, incubation times, application dynamics, post-mortem, followed by removal of the entire wound bed wound bed structure, associated disease state (e.g., diabetes, (e.g., with underlying stromal muscular elements). Similar to etc.) and the like that make each wound bed unique. As such, the model in vitro system as previously described, the ex vivo the present invention provides for the alteration of surface model system includes linker chemistries and the like, and the chemistry/structure for each unique wound bed, using the immobilization strategies are evaluated, for example, using compositions and methods as described herein. fluorescently labelled proteins. In some embodiments, the ex vivo model system comprises the immobilization of biotiny EXPERIMENTAL lated BSA. In some embodiments, the surface of the wound 0265. The examples below serve to further illustrate the bed is activated with a NHS ester by exposureto, for example, invention, to provide those of ordinary skill in the art with a 1 mMBS. Biotinylated BSA (at, for example, 2 mg/ml) is complete disclosure and description of how the compounds, subsequently added to the activated wound bed and allowed compositions, articles, devices, and/or methods claimed to attach for 2 hours. The biotinylated BSA immobilization is herein are made and evaluated, and are not intended to limit determined by FITC-labeled anti-biotin antibody. As such, the scope of the invention. The examples are not intended to once immobilization is verified (via fluorescence detection), restrict the scope of the invention. evaluation of optimal chemical functionalities is determined for wound bed immobilization. For example, chemical func EXAMPLE 1. tionalities are determined by replacing BS in the ex vivo Covalent Immobilization of a Protein to a Surface model system with heterobifunctional cross-linkers as 0266 To demonstrate that proteins can be covalently described herein that react with, for example, carboxylic immobilized on model surfaces, bovine serum albumin US 2011/O 1892.87 A1 Aug. 4, 2011
(BSA) was used as the protein in the model system. Bovine Cutaneous wounds from euthanized mice are harvested. The serum albumin was biotinylated (BSA used as control) and excised wounds are sequentially treated by the spraying (us covalently attached to gamma-amino propylsilanes (GAPS)- ing aerosol spray cans) of aqueous solutions (0.5M NaCl, treated glass Surfaces using the homobifunctional bifunc PBS at pH 7.0) containing polystyrene sulfonate (PSS, 1 tional cross-linker BS (1 mM for 15 min.). BS contains an mg/ml) or FITC-labeled poly allylamine hydrochloride amine-reactive N-hydroxysulfosuccinimide (NHS) ester at (FITC-PAH, 1 mg/ml). Between spraying steps, the wounds each end of an 8-carbon spacer arm. The NHS esters react are treated rinsed with PBS. After each treatment with FITC with primary amines at pH 7-9 to form stable amide bonds, PAH, the intensity of fluorescence is recorded. A measure along with release of the N-hydroxysulfosuccinimide leaving ment of the growth in fluorescence after each treatment cycle group. For detection purposes, the biotinylated BSA was with PSS and FITC-PAH demonstrates growth of a multilay labeled with FITC-labeled anti-biotinantibodies and the BSA ered polyelectrolyte film on the wound bed, relative to control control was probed using FITC-antigoat antibodies. treatmentS. 0267. The large fluorescent signal seen in FIG.3 from the surfaces treated with biotinylated BSA and FITC-antibiotin EXAMPLE 5 antibody (diamonds) relative to controls (vertical lines, diagonal lines, bricks) confirms covalent attachment of the Delivery of the Wound Modifying Agents using biotinylated BSA to the surface. Pumps 0271 Experiments depositing polyelectrolytes in wound EXAMPLE 2 bed explants from mice are performed by sequentially deliv Layer by Layer Deposition of Polyelectrolytes in ex ering the wound healing agent to the wound beds using vivo Wound Beds pumps. Cutaneous wounds from euthanized mice are har Vested. A peristaltic pump (Fischer Scientific) is used to pump 0268 Experiments depositing polyelectrolytes in wound the liquid. The excised wounds are sequentially treated by bed explants from mice were performed. Cutaneous wounds pumping of aqueous solutions (0.5M NaCl, PBS at pH 7.0) from euthanized mice were harvested. The excised wounds containing polystyrene sulfonate (PSS, 1 mg/ml) or FITC were sequentially treated with aqueous solutions (0.5M labeled poly allylamine hydrochloride (FITC-PAH, 1 NaCl, PBS at pH 7.0) containing polystyrene sulfonate (PSS, mg/ml). Between pumping steps involving the polyelectro 1 mg/ml) and FITC-labeled poly allylamine hydrochloride lytes, the wounds are treated rinsed with PBS using pumps. (FITC-PAH, 1 mg/ml). Between adsorption steps, the After each treatment with FITC-PAH, the intensity of fluo wounds were treated rinsed with PBS. After each treatment rescence is recorded. A measurement of the growth in fluo with FITC-PAH, the intensity of fluorescence was recorded. rescence after each treatment cycle with PSS and FITC-PAH As seen in FIG. 4, the growth in fluorescence after each demonstrates growth of a multilayered polyelectrolyte film treatment cycle with PSS and FITC-PAH demonstrates on the wound bed, relative to control treatments. growth of a multilayered polyelectrolyte film on the wound bed, relative to control treatments. EXAMPLE 6 Delivery of the Wound Modifying Agents using EXAMPLE 3 Stamps Use of Mesoscopic Cross-Linkers in ex vivo Wound 0272 Experiments depositing polyelectrolytes in wound Beds bed explants from mice are performed by stamping of poly 0269. Skin wounds are created in tandem on diabetic mice electrolytes into the wound bed from elastomeric stamps. using a Surgical skin punch. After wounding, one of the Elastomeric stamps are prepared from PDMS using parts A wounds is treated by contact with activated 10-micrometer and B of a kit purchased from Dow-Corning. After curing the diameter polystyrene beads with Surfaces terminated in car stamps in an oven at 100 oC for 12 hrs, the surface of the boxylic acid groups. The activation of the carboxylic acid PDMS stamp is incubated with aqueous solutions (0.5M groups is achieved by incubation in EDC/NHS solution (200 NaCl, PBS at pH 7.0) containing polystyrene sulfonate (PSS, mM/50 mM) in phosphate buffered saline (PBS) (10 mM 1 mg/ml) or FITC-labeled poly allylamine hydrochloride phosphate, 120 mM. NaCl, 2.7 mM KCl; pH 7.6) for 1 h at (FITC-PAH, 1 mg/ml). Between incubation steps involving room temperature. Following activation, the beads were the polyelectrolytes, the PDMS stamp is rinsed with PBS. pipetted into the wound beads and allowed to incubate within After 10 layers each of PSS and PAH are deposited onto the the wound beds for 1 hr. After incubation, the wound beds are stamp, the stamp is mechanically contacted with the cutane washed exhaustively with PBS, and then incubated with col ous wounds harvested from euthanized mice. After removal lagen (10 micromolar in PBS). The remaining wound is used of the stamp from the wound bed, the intensity of fluores as a control and treated as described above, but without acti cence of the wound bed is recorded. A measurement of the vation of the beads with NHS/EDC. The size of the wound is fluorescence relative to control treatments (PBS free of poly measured after 2, 4, 6, 8 and 10 days. It is observed that the electrolytes is incubated with the stamps) verifies the depo treated wound decreases in size faster than the control wound. sition of the wound modifying agent into the wound bed. EXAMPLE 4 EXAMPLE 7 Delivery of the Wound Modifying Agents using Delivery of the Wound Modifying Agents to Sur Aerosol Sprays faces using Inkjet Technology 0270 Experiments depositing polyelectrolytes in wound 0273 To demonstrate that inkjet technology can be used to bed explants from mice are performed using aerosol sprays. deliver wound modifying agents to Surfaces, aqueous solu US 2011/O 1892.87 A1 Aug. 4, 2011 42 tions (in PBS) of FITC-labelled collagen are inkjet printed wounds is treated by contact with activated 1-micrometer onto gamma-amino propyl silanes (GAPS)-treated glass Sur diameter polystyrene beads with Surfaces terminated in car faces that are activated using the homobifunctional bifunc boxylic acid groups. The activation of the carboxylic acid tional cross-linker BS (1 mM for 15 min.). BS contains an groups is achieved by incubation in EDC/NHS solution (200 amine-reactive N-hydroxysulfosuccinimide (NHS) ester at mM/50 mM) in phosphate buffered saline (PBS) (10 mM each end of an 8-carbon spacer arm. The NHS esters react phosphate, 120 mM. NaCl, 2.7 mM KCl; pH 7.6) for 1 hour at with primary amines at pH 7-9 to form stable amide bonds, room temperature. Following activation, the beads are pipet along with release of the N-hydroxysulfosuccinimide leaving ted into the wound beds and allowed to incubate within the group. The surfaces are exhaustively washed with PBS. The wound beds for 1 hr. After incubation, the wound beds are fluorescence intensity of the Surface is measurements to indi washed exhaustively with PBS, and then incubated with PAH, cate attachment of the FITC-labelled collagen to the surface and then washed again in PBS. After PAH, the wound bed is relative to a control experiment in which the activated surface sequentially treated with PSS and FITC-labelled PAH (as is pre-exposed to BSA (1 mg/ml in PBS for 2 hrs), prior to described above). Between each adsorption step, the wound inject printing of the FITC-labelled collagen. bed is rinsed with PBS. Fluorescence measurements of the wound bed after each FITC-PAH adsorption step confirm EXAMPLE 8 immobilization of the PSS and PAH to the wound bed. Delivery of the Wound Modifying Agents using Air Brushes EXAMPLE 11 0274 Experiments depositing polyelectrolytes in wound Method to Modify the Mechanical Compliance of bed explants from mice are performed using airbrushes. Cuta the Wound Bed neous wounds from euthanized mice are harvested. The excised wounds are sequentially treated by the airbrushing of 0277. The mechanical compliance of the wound bed is aqueous solutions (0.5M NaCl, PBS at pH 7.0) containing modified by depositing polyelectrolytes into the wound bed polystyrene sulfonate (PSS, 1 mg/ml) or FITC-labeled poly and cross-linking the polyelectrolyte films. Experiments allylamine hydrochloride (FITC-PAH, 1 mg/ml). The air depositing polyelectrolytes in woundbed explants from mice brushes are prepared by pouring the aqueous polyelectrolyte are performed. Cutaneous wounds from euthanized mice are solutions into the air brushes. Between brushing steps, the harvested. The excised wounds are sequentially treated with wounds are rinsed with PBS. After each treatment with FITC aqueous solutions (0.5M NaCl, PBS at pH 7.0) containing PAH, the intensity of fluorescence is recorded. A measure polystyrene sulfonate (PSS, 1 mg/ml) and FITC-labeled poly ment of the growth in fluorescence after each treatment cycle allylamine hydrochloride (FITC-PAH, 1 mg/ml). Between with PSS and FITC-PAH demonstrates growth of a multilay adsorption steps, the wounds are treated and rinsed with PBS. ered polyelectrolyte film on the wound bed, relative to control After treatment with the polyelectrolytes, BS3 (1 mMinPBS) is added to the wound bed and incubated for 1 hr. Upon treatments in which PBS solutions free of polyelectrolytes are poking with the end of a spatula, the rigidity of the woundbed airbrushed onto the surfaces. is noticeably greater than a wound bed treated with polyelec EXAMPLE 9 trolytes without the final cross-linking step. The increase in Method to Modify the Topography of the Wound ridigidity (decreased compliance) is confirmed by Atomic Bed Force Microscopy. 0275 Skin wounds are created in tandem on diabetic mice EXAMPLE 12 using a Surgical skin punch. After wounding, one of the wounds is treated by contact with a 1:1 mixture of activated Method to Alter Intrinsic Compliance of Wound Bed 100-nanometer and 1 micrometer diameter polystyrene beads 0278. The mechanical compliance of the wound bed is with Surfaces terminated in carboxylic acid groups. The acti altered by controlled application of enzyme(s) capable of Vation of the carboxylic acid groups is achieved by incubation degrading constituents of the extracellular matrix. Cutaneous in EDC/NHS solution (200 mM/50 mM) in phosphate buff wounds from euthanized mice are harvested. 10 uM concen ered saline (PBS) (10 mM phosphate, 120 mMNaCl, 2.7 mM tration of collagenase is applied to the wound and allowed to KCl; pH 7.6) for 1 hour at room temperature. Following incubate at room temperature for times ranging from 0 min-1 activation, the mixture of beads are pipetted into the wound hour. The wound beds are then rinsed copiously with PBS. beads and allowed to incubate within the woundbeds for 1 hr. Wet Field force microscopy of the wound beds confirms that After incubation, the wound beds are washed exhaustively application of degradative enzymes increases the compliance with PBS, and then incubated in albumin (10 micromolar in (decreases rigidity) of the wound beds in a time dependent PBS). The remaining wound is used as a control and treated as fashion. described above, but without activation of the beads with NHS/EDC. The size of the wound is measured after 2, 4, 6, 8 EXAMPLE 13 and 10 days. It is observed that the treated wound decreases in size faster than the control wound. Nanometer-Thick Silver-Impregnated Polymeric Films Showing Selective Toxicity Towards Bacterial EXAMPLE 10 Growth and that Support Mammalian Cell Growth Method to Covalently Modify the Wound Bed using (0279 Polyelectrolyte multilayers electrostatically a Combination of Polyelectrolytes and Covalent assembled alternately from Such weak polyacids as poly(al Cross-Linking Agents lylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) 0276 Skin wounds are created in tandem on diabetic mice were tuned to impregnate post-assembly with silver nanopar using a Surgical skin punch. After wounding, one of the ticles to levels where they kill bacteria effectively yet allow US 2011/O 1892.87 A1 Aug. 4, 2011
adhesion of mammaliancells on the films without measurable What is claimed is: cytotoxicity. Silver ions (Ag") were incorporated in the films 1. An article comprising: by incubating them with bulk solution of a silver salt (e.g., a polymer multilayer from about 1 nm to 500 nm thick silver nitrate). While the present invention is not limited to comprising at least one polymer, said polymer multi any particular mechanism, and an understanding of the layer having associated therewith particles selected mechanism is not necessary to practice the present invention, from the group consisting of nanoscale and microscale it is contemplated that Ag" ions bound to the free carboxylic particles. acid groups of PAA in the films by ion exchange with the 2. The article of claim 1, wherein said at least one polymer acidic protons. Post binding, Ag" in the films were reduced to is selected from the group consisting of a cationic polymer, an Zerovalent Ag nanoparticles by using an aqueous solution of anionic polymer, a nonionic polymeran amphoteric polymer a reducing agent. By changing the pH of the polyelectrolyte and combinations thereof assembly solutions, the number of non-ionized carboxylic 3. The article of claim 1, wherein the diameter of said acid groups (and hence the amount of silver incorporated) in particles is greater than the thickness of the polymer multi the PEMs post-assembly was effectively controlled. These layer. silver-impregnated nanometer-thick films were then investi 4. The article of any of claim 1, further comprising at least gated for their interactions with NIH-3T3 cells, a highly adhe one antimicrobial agent. sive murine fibroblast cell line, and Staphylococcus epider 5. The article of claim 4, wherein aid at least one antimi midis, a gram-positive bacterium that occurs frequently on crobial agent is an antimicrobial silver composition. skin and in mucous membranes. 6. The article of claim 5, wherein said antimicrobial silver 0280 Silver loading in the PEMs could be varied from composition is provided in an amount so that said antimicro ~24.2 ug/cm to 0.4 ug/cm. Cytotoxicity to NIH-3T3 cells bial silver composition is released at a rate of about 0.01 and seeded on these films was correlated with the concentration of 100 82 g/cm per day. soluble silver in the PEMs, with augmented toxicity at higher 7. The article of claim 1, wherein said particles are associ silver concentrations, as shown in FIG. 8. PEMs prepared ated with said polymer multilayer in a manner selected from with relatively more ionized PAA entrapped lower amounts the group consisting of interspersed or dispersed in said poly of silver and demonstrated reduced cytotoxicity. Further, mer multilayer, displayed on a surface of said polymer mul their strongly ionically crosslinked architecture allowed bet tilayer; and underneath the polymer multilayer. ter attachment and spreading of fibroblasts than PEMs pre 8. The article of claim 1, wherein said polymer multilayer pared using weakly ionized PAA, as shown in FIG. 10. Bac is disposed on a support. teria killing efficiency of PEMs correlated to the amount of 9. The article of claim 8, wherein said support is selected silver entrapped in the films (FIG. 9), with PEMs containing from the group consisting of a polymeric Support, an elasto as little as -0.4 g/cm of silver providing up to 99.9999%kill meric Support, a silicone Support, a fabric Support, a wound in a water-borne assay. Furthermore, PEMs with this silver dressing and a biologic wound dressing. loading did not exhibit measurable cytotoxicity to NIH-3T3 10. The article of claim 8, wherein said support comprises cells. a material selected from the group consisting of an absorbent material, a foam, a transparent thin film, polyurethane thin films, a hydrogel, hydrofibers, hydrocolloids, alginate fibers, EXAMPLE 1.4 silica gel, Sodium polyacrylate, potassium polyacrylamides Transfer to a Soft Surface with Microscale Beads and combinations thereof. 11. The article of claims 8, wherein said support comprises 0281 Introducing rigid microspheres in the polymer mul a biological molecule selected from the group consisting of a tilayers facilitates their transfer onto the dermis layer of a biological molecule selected from the group consisting of commercially available human skin graft-GammaCraft(R). A collagen, hyaluronic acid, glycosaminoglycans, keratin, PAH(PAA/PAH)10 multilayer, with fluorescent PAH and a fibronectin, vitronectin, laminin, and combinations or frag PAH(PAA/PAH)5(PS u-spheres)(PAH/PAA)5PAH multi ments thereof layer, with fluorescent PS microspheres were stamped onto 12. The article of claim 8, wherein said support comprises skin grafts. The portion of the multilayers that adhered to the cells or a tissue matrix. grafts were accessed by fluorescence microscopy and imag 13. The article of claim 8, wherein said support and said ing. The results (FIG. 12) showed that the polymer multilay polymer multilayer are separated by a water soluble material. ers with microscale beads showed much better transfer to the 14. The article of claim 8, wherein said support comprises skin graft than did the polymer multilayer without microscale a first Surface comprising an anti-adherent material. beads. 15. The article of claim 14, wherein said polymer multi 0282 All publications and patents mentioned in the above layer is deposited on said first Surface comprising an anti specification are herein incorporated by reference. Various adherent material. modifications and variations of the described method and 16. The article of claim 15, wherein said absorbent material system of the invention will be apparent to those skilled in the comprises a second Surface fixed to an adhesive material. art without departing from the scope and spirit of the inven 17. The article of claim 8, wherein said polymer multilayer tion. Although the invention has been described in connection is deposited onto said support by a method selected from the with specific preferred embodiments, it should be understood group consisting of adsorption from Solution, spraying, and that the invention as claimed should not be unduly limited to spin coating. Such specific embodiments. Indeed, various modifications of 18. The article of claim 1, wherein said article is packaged the described modes for carrying out the invention that are in a sterile package. obvious to those skilled in relevant fields are intended to be 19. The article of claim 1, wherein said article does not within the scope of the following claims. Substantially impair the healing of wounds. US 2011/O 1892.87 A1 Aug. 4, 2011 44
20. An article comprising: 24. A method comprising contacting a surface with a poly a polymer multilayer having a releasable antimicrobial mer multilayer comprising at least one polymer, said polymer silver composition incorporated therein, wherein said multilayer having associated therewith particles selected releasable antimicrobial silver composition is releasable from the group consisting of nanoscale and microscale par in an amount of about 0.01 and 100 ug/cm of said ticles under conditions such that said polymer multilayer is polymer multilayer per day. transferred to said surface. 21. An article comprising: 25. The method of claim 24, wherein said surface is selected from the group consisting of skin, a wound bed, a a solid Support; and tissue, an artificial tissue matrix, a wound dressing, and a a polymer multilayer Supported by said solid Support, said biologic wound dressing. polymer multilayer comprising at least one wound 26. The method of claim 24, whereintransfer is done in the active agent. Substantial absence of a solution or a liquid solvent. 22. The article of claim 21, wherein said wound active 27. The method of claim 24, wherein said transfer is a dry agent is selected from the group consisting of analgesics, transfer. extracellular matrix components, growth factors, honey, and 28. A method of treating a subject having a wound com wound debriding agents. prising: 23. An article comprising: providing an article according to claim 1 and contacting a a solid Support comprising a releasable antimicrobial silver wound on said subject with said article. composition, wherein said releasable antimicrobial sil 29. A kit comprising the article of claim 1. vercomposition is releasable in an amount of about 0.01 and 100 ug/cm of said polymer multilayer per day. c c c c c