US20220273845A1 - Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures - Google Patents
Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures Download PDFInfo
- Publication number
- US20220273845A1 US20220273845A1 US17/740,071 US202217740071A US2022273845A1 US 20220273845 A1 US20220273845 A1 US 20220273845A1 US 202217740071 A US202217740071 A US 202217740071A US 2022273845 A1 US2022273845 A1 US 2022273845A1
- Authority
- US
- United States
- Prior art keywords
- cardiac
- gold
- peptides
- cell
- gold nanostructures
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 66
- 239000010931 gold Substances 0.000 title claims abstract description 58
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 57
- 238000005034 decoration Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 36
- 210000004165 myocardium Anatomy 0.000 claims abstract description 35
- 239000000203 mixture Substances 0.000 claims abstract description 24
- 230000008929 regeneration Effects 0.000 claims abstract description 11
- 238000011069 regeneration method Methods 0.000 claims abstract description 11
- 241001465754 Metazoa Species 0.000 claims abstract description 10
- 230000008439 repair process Effects 0.000 claims abstract description 9
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 61
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 39
- 210000004413 cardiac myocyte Anatomy 0.000 claims description 38
- 125000005647 linker group Chemical group 0.000 claims description 29
- 230000021164 cell adhesion Effects 0.000 claims description 25
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical class NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 claims description 19
- 230000000982 vasogenic effect Effects 0.000 claims description 15
- 125000004432 carbon atom Chemical group C* 0.000 claims description 13
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 12
- 210000000803 cardiac myoblast Anatomy 0.000 claims description 12
- 210000002950 fibroblast Anatomy 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 10
- 125000001183 hydrocarbyl group Chemical group 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- -1 polyethyleneoxy chain Polymers 0.000 claims description 10
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 claims description 5
- 125000004454 (C1-C6) alkoxycarbonyl group Chemical group 0.000 claims description 5
- 125000006700 (C1-C6) alkylthio group Chemical group 0.000 claims description 5
- 125000005862 (C1-C6)alkanoyl group Chemical group 0.000 claims description 5
- 125000005913 (C3-C6) cycloalkyl group Chemical group 0.000 claims description 5
- 125000004423 acyloxy group Chemical group 0.000 claims description 5
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 5
- 125000003118 aryl group Chemical group 0.000 claims description 5
- 125000004104 aryloxy group Chemical group 0.000 claims description 5
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 5
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 5
- 125000001072 heteroaryl group Chemical group 0.000 claims description 5
- 125000005553 heteroaryloxy group Chemical group 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 5
- 125000004043 oxo group Chemical group O=* 0.000 claims description 5
- 125000001424 substituent group Chemical group 0.000 claims description 5
- 230000002424 anti-apoptotic effect Effects 0.000 claims description 4
- 230000000421 anti-necrotic effect Effects 0.000 claims description 4
- HZHXMUPSBUKRBW-FXQIFTODSA-N (4s)-4-[[2-[[(2s)-2-amino-3-carboxypropanoyl]amino]acetyl]amino]-5-[[(1s)-1-carboxyethyl]amino]-5-oxopentanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O HZHXMUPSBUKRBW-FXQIFTODSA-N 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 108010053299 glycyl-arginyl-glycyl-aspartyl-seryl-proline Proteins 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 230000008685 targeting Effects 0.000 claims description 3
- 108700011259 MicroRNAs Proteins 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- 210000004263 induced pluripotent stem cell Anatomy 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- NTEDOEBWPRVVSG-FQUUOJAGSA-N (2s)-1-[(2r)-2-[[(2s)-2-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carboxylic acid Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CO)C(=O)N1CCC[C@H]1C(O)=O NTEDOEBWPRVVSG-FQUUOJAGSA-N 0.000 claims 1
- 239000003963 antioxidant agent Substances 0.000 claims 1
- 230000003078 antioxidant effect Effects 0.000 claims 1
- 125000001475 halogen functional group Chemical group 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 40
- 230000000747 cardiac effect Effects 0.000 description 30
- 210000001054 cardiac fibroblast Anatomy 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 23
- 230000015572 biosynthetic process Effects 0.000 description 20
- 238000013459 approach Methods 0.000 description 16
- 230000008878 coupling Effects 0.000 description 13
- 238000010168 coupling process Methods 0.000 description 13
- 238000005859 coupling reaction Methods 0.000 description 13
- 238000003501 co-culture Methods 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 108010010803 Gelatin Proteins 0.000 description 9
- 230000001413 cellular effect Effects 0.000 description 9
- 239000008273 gelatin Substances 0.000 description 9
- 229920000159 gelatin Polymers 0.000 description 9
- 235000019322 gelatine Nutrition 0.000 description 9
- 235000011852 gelatine desserts Nutrition 0.000 description 9
- 230000010354 integration Effects 0.000 description 9
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 230000002792 vascular Effects 0.000 description 8
- 102000001045 Connexin 43 Human genes 0.000 description 7
- 108010069241 Connexin 43 Proteins 0.000 description 7
- 239000012620 biological material Substances 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000000017 hydrogel Substances 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 6
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 230000021615 conjugation Effects 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
- 230000001360 synchronised effect Effects 0.000 description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 5
- 102000010970 Connexin Human genes 0.000 description 5
- 108050001175 Connexin Proteins 0.000 description 5
- 102100030540 Gap junction alpha-5 protein Human genes 0.000 description 5
- 239000011575 calcium Substances 0.000 description 5
- 229910052791 calcium Inorganic materials 0.000 description 5
- 239000002041 carbon nanotube Substances 0.000 description 5
- 210000002744 extracellular matrix Anatomy 0.000 description 5
- 238000007306 functionalization reaction Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000010899 nucleation Methods 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102100039290 Gap junction gamma-1 protein Human genes 0.000 description 4
- 206010019280 Heart failures Diseases 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 229910021393 carbon nanotube Inorganic materials 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 230000008602 contraction Effects 0.000 description 4
- 239000004205 dimethyl polysiloxane Substances 0.000 description 4
- 125000005843 halogen group Chemical group 0.000 description 4
- 210000005003 heart tissue Anatomy 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 210000000107 myocyte Anatomy 0.000 description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 210000002235 sarcomere Anatomy 0.000 description 4
- 229930195734 saturated hydrocarbon Natural products 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 102000016359 Fibronectins Human genes 0.000 description 3
- 108010067306 Fibronectins Proteins 0.000 description 3
- 206010029113 Neovascularisation Diseases 0.000 description 3
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- 238000003917 TEM image Methods 0.000 description 3
- 230000036982 action potential Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 230000008619 cell matrix interaction Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 210000002064 heart cell Anatomy 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 230000003278 mimic effect Effects 0.000 description 3
- 208000010125 myocardial infarction Diseases 0.000 description 3
- 239000002055 nanoplate Substances 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 230000006320 pegylation Effects 0.000 description 3
- 229910052710 silicon Inorganic materials 0.000 description 3
- 239000010703 silicon Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 230000002861 ventricular Effects 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- NTEDOEBWPRVVSG-XUXIUFHCSA-N (2s)-1-[(2s)-2-[[(2s)-2-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carboxylic acid Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(O)=O NTEDOEBWPRVVSG-XUXIUFHCSA-N 0.000 description 2
- 102000010825 Actinin Human genes 0.000 description 2
- 108010063503 Actinin Proteins 0.000 description 2
- 101800000068 Antioxidant peptide Proteins 0.000 description 2
- 101710177922 Gap junction alpha-5 protein Proteins 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 238000001237 Raman spectrum Methods 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 238000010908 decantation Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000005395 methacrylic acid group Chemical group 0.000 description 2
- 238000009343 monoculture Methods 0.000 description 2
- 230000010016 myocardial function Effects 0.000 description 2
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000003076 paracrine Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 210000003742 purkinje fiber Anatomy 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 238000011808 rodent model Methods 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- RPENMORRBUTCPR-UHFFFAOYSA-M sodium;1-hydroxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].ON1C(=O)CC(S([O-])(=O)=O)C1=O RPENMORRBUTCPR-UHFFFAOYSA-M 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 description 1
- OBLNMUKJWUIHTD-JYJNAYRXSA-N (3s)-3-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-[[(1s)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]-4-oxobutanoic acid Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OBLNMUKJWUIHTD-JYJNAYRXSA-N 0.000 description 1
- MZDFTCYQDDMLON-UHFFFAOYSA-N (4-amino-4-oxobutanoyl)-hydroxysulfamic acid Chemical compound NC(=O)CCC(=O)N(O)S(O)(=O)=O MZDFTCYQDDMLON-UHFFFAOYSA-N 0.000 description 1
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
- RQEUFEKYXDPUSK-UHFFFAOYSA-N 1-phenylethylamine Chemical compound CC(N)C1=CC=CC=C1 RQEUFEKYXDPUSK-UHFFFAOYSA-N 0.000 description 1
- TXHAHOVNFDVCCC-UHFFFAOYSA-N 2-(tert-butylazaniumyl)acetate Chemical compound CC(C)(C)NCC(O)=O TXHAHOVNFDVCCC-UHFFFAOYSA-N 0.000 description 1
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 1
- WNYJVAMZRBTOPE-YVSFHVDLSA-N 2-[(2s,5r,8r,11s)-5-benzyl-11-[3-(diaminomethylideneamino)propyl]-3,6,9,12,15-pentaoxo-8-(sulfanylmethyl)-1,4,7,10,13-pentazacyclopentadec-2-yl]acetic acid Chemical compound N1C(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)NC(=O)[C@H]1CC1=CC=CC=C1 WNYJVAMZRBTOPE-YVSFHVDLSA-N 0.000 description 1
- BXRLWGXPSRYJDZ-UHFFFAOYSA-N 3-cyanoalanine Chemical compound OC(=O)C(N)CC#N BXRLWGXPSRYJDZ-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 102000008076 Angiogenic Proteins Human genes 0.000 description 1
- 108010074415 Angiogenic Proteins Proteins 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 102000014814 CACNA1C Human genes 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 208000005422 Foreign-Body reaction Diseases 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 101710178004 Gap junction gamma-1 protein Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 101000867811 Homo sapiens Voltage-dependent L-type calcium channel subunit alpha-1C Proteins 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102000012355 Integrin beta1 Human genes 0.000 description 1
- 108010022222 Integrin beta1 Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- DGYHPLMPMRKMPD-UHFFFAOYSA-N L-propargyl glycine Natural products OC(=O)C(N)CC#C DGYHPLMPMRKMPD-UHFFFAOYSA-N 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 102100026057 Myosin regulatory light chain 2, atrial isoform Human genes 0.000 description 1
- 101710098224 Myosin regulatory light chain 2, atrial isoform Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- BZQFBWGGLXLEPQ-UHFFFAOYSA-N O-phosphoryl-L-serine Natural products OC(=O)C(N)COP(O)(O)=O BZQFBWGGLXLEPQ-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000004912 RYR2 Human genes 0.000 description 1
- 108060007241 RYR2 Proteins 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 229920001486 SU-8 photoresist Polymers 0.000 description 1
- 102100027732 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 Human genes 0.000 description 1
- 101710109123 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- QOMNQGZXFYNBNG-UHFFFAOYSA-N acetyloxymethyl 2-[2-[2-[5-[3-(acetyloxymethoxy)-2,7-difluoro-6-oxoxanthen-9-yl]-2-[bis[2-(acetyloxymethoxy)-2-oxoethyl]amino]phenoxy]ethoxy]-n-[2-(acetyloxymethoxy)-2-oxoethyl]-4-methylanilino]acetate Chemical compound CC(=O)OCOC(=O)CN(CC(=O)OCOC(C)=O)C1=CC=C(C)C=C1OCCOC1=CC(C2=C3C=C(F)C(=O)C=C3OC3=CC(OCOC(C)=O)=C(F)C=C32)=CC=C1N(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O QOMNQGZXFYNBNG-UHFFFAOYSA-N 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 108010027234 aspartyl-glycyl-glutamyl-alanine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000002051 biphasic effect Effects 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- UHBYWPGGCSDKFX-UHFFFAOYSA-N carboxyglutamic acid Chemical compound OC(=O)C(N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-UHFFFAOYSA-N 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 229960000800 cetrimonium bromide Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 108010086416 cyclo(arginyl-glycyl-aspartyl-phenylalanyl-cysteinyl) Proteins 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000010217 densitometric analysis Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 229950006137 dexfosfoserine Drugs 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002296 dynamic light scattering Methods 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000000806 elastomer Substances 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- YAGKRVSRTSUGEY-UHFFFAOYSA-N ferricyanide Chemical compound [Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-N 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 108010057753 glycyl-arginyl-glycyl-aspartyl-tyrosine Proteins 0.000 description 1
- 150000002343 gold Chemical class 0.000 description 1
- 229910021389 graphene Inorganic materials 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000031852 maintenance of location in cell Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000013160 medical therapy Methods 0.000 description 1
- 230000005499 meniscus Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000693 micelle Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 239000002073 nanorod Substances 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 239000002070 nanowire Substances 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- CQYBNXGHMBNGCG-RNJXMRFFSA-N octahydroindole-2-carboxylic acid Chemical compound C1CCC[C@H]2N[C@H](C(=O)O)C[C@@H]21 CQYBNXGHMBNGCG-RNJXMRFFSA-N 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 1
- 239000012285 osmium tetroxide Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229960001639 penicillamine Drugs 0.000 description 1
- 125000001151 peptidyl group Chemical group 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920001992 poloxamer 407 Polymers 0.000 description 1
- 229920003213 poly(N-isopropyl acrylamide) Polymers 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 230000004537 potential cytotoxicity Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 210000004683 skeletal myoblast Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000002174 soft lithography Methods 0.000 description 1
- DFVFTMTWCUHJBL-BQBZGAKWSA-N statine Chemical compound CC(C)C[C@H](N)[C@@H](O)CC(O)=O DFVFTMTWCUHJBL-BQBZGAKWSA-N 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 230000006459 vascular development Effects 0.000 description 1
- 238000010865 video microscopy Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/047—Other specific metals or alloys not covered by A61L27/042 - A61L27/045 or A61L27/06
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3826—Muscle cells, e.g. smooth muscle cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3873—Muscle tissue, e.g. sphincter
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/165—Vascular endothelial growth factor [VEGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/10—Mineral substrates
Definitions
- CMs cardiomyocytes
- Heart transplantation or implantation of mechanical left ventricular assist device are treatment options of last resort, but are limited by inadequate organ donors and potential complications of the surgical procedures.
- treatment is limited to pharmacologic therapy to optimize the function of remaining CMs and slow the adverse cardiac remodeling.
- MRT myocardial replacement therapy
- Cell-based MRT involves local injection of stem/progenitor or adult cells into infarcted myocardium to augment existing pool of CMs and initiate an endogenous regeneration process. This approach has many potential advantages including use of less invasive procedures, lack of reliance on availability of donor hearts and use of patient's own cells to obviate immune reactions.
- Cell-based MRT approaches include of direct injection of adult cells (e.g. skeletal myoblasts, mesenchymal), cardiac stem/progenitor cells (e.g.
- Current cell-based MRT approaches are hampered by poor ( ⁇ 50%) cell retention, inadequate cell-cell coupling and poor engraftment with the host myocardium, with only 10% survival of injected cells.
- Various approaches utilized natural (e.g. alginate, fibrin, collagen, extracellular matrices) or synthetic materials (i.e.
- PNIPAAm self-assembling peptides
- injection or implantation i.e. patch
- PNIPAAm self-assembling peptides
- injection or implantation i.e. patch
- cell-embedded biomaterials have so far demonstrated inconsistent outcomes due to lack of electromechanical integration (i.e. synchronous contraction) of the tissues with the host myocardium, raising the potential risk of arrhythmias, as well as poor vascularization.
- implantation of cell-embedded patches can potentially cause immune rejection or foreign body reactions while requiring a highly invasive approach (i.e. open-heart surgery) to implant the tissues.
- Native ventricular myocardium consists of CMs coupled with electrically conductive purkinje fibers and mechanically robust extracellular matrix (ECM). This unique architecture exhibits tightly packed and aligned (i.e. anisotropic) cellular constructs. Following injury, replacement of infarcted myocardium would require proper scaffolding biomaterials and cell sources to mimic the structural architecture of native myocardium.
- CNT Carbon nanotubes
- a scaffold-free microtissue comprising gold nanostructures within the microtissue.
- the microtissue comprises cardiac myocytes, endothelial cells, pluripotent stem cells, myoblasts, or fibroblasts.
- the gold nanostructures are 1D gold nanostructures.
- the 1D gold nanostructures are wires, rods, or spheres.
- the gold nanostructures include 2D gold nanostructures.
- the 2D gold nanostructures are nano-plates.
- one or more gold nanostructures are linked to a cell adhesion moiety.
- the cell adhesion moiety is a peptide.
- a cardiac microtissue comprising nanoscale decoration of cardiac cells including gold nanostructures within the microtissue.
- the microtissue is scaffold free.
- the gold nanostructures are functionalized using RGD cell adhesion motifs.
- the gold nanostructures are capped with polyethylene glycol bi-linker.
- the microtissue is spheroid based.
- the gold nanostructures are conjugated with vasculogenic peptides.
- the microtissue further includes functionalized cardiomyocytes and cardiac fibroblasts within the gold nanostructures.
- an electrically conductive composition comprising cardiac micro-tissue and gold nanostructures is described herein.
- a method for regeneration or repair of infarcted myocardium in an animal comprises injecting a composition as described above.
- the composition is injected within the infarcted region of myocardium of the animal.
- an electrically conductive composition as described above for use in medical therapy is provided.
- an electrically conductive composition as described above for regeneration or repair of infarcted myocardium in one or more embodiments, an electrically conductive composition as described above for regeneration or repair of infarcted myocardium.
- an electrically conductive composition as described above to prepare a medicament for regeneration or repair of infarcted myocardium.
- a scaffold-free microtissue comprises one or more gold nanostructures linked to a functional moiety, wherein the functional moiety is one or more vasculogenic peptides, one or more anti-inflammatory peptides, one or more antiapoptotic peptides, one or more antinecrotic peptides, one or more antioxidant peptides, one or more oligonucleotides, one or more lipid particles, one or more phospholipid particles, one or more liposomes, one or more nanoliposomes, one or more microRNAs, or one or more siRNAs.
- the functional moiety is one or more vasculogenic peptides, one or more anti-inflammatory peptides, one or more antiapoptotic peptides, one or more antinecrotic peptides, one or more antioxidant peptides, one or more oligonucleotides, one or more lipid particles, one or more phospholipid particles, one or more liposomes, one or more nanoliposomes
- the scaffold-free microtissue further comprises a plurality of cardiac myocytes or cardiac myoblasts, wherein the cardiac myocytes or cardiac myoblasts are conjugated to the one or more gold nanostructures, wherein the plurality of cardiac myocytes or cardiac myoblasts are arranged in a cluster.
- the scaffold-free microtissue further comprises a plurality of fibroblasts, wherein the fibroblasts are arranged in at least one layer of fibroblasts that substantially surrounds the cluster of gold-nanostructure-conjugated cardiac myocytes or gold-nanostructure-conjugated cardiac myoblasts.
- a method for regeneration or repair of an infarcted myocardium including an infarcted region in an animal comprises injecting a scaffold-free microtissue into the animal.
- FIG. 1A illustrates conjugated, scaffold-free cardiac micro tissues using electrically conductive, cell adhesion-promoting and vasculogenic gold nanowires (GNWs), in accordance with one or more embodiments.
- GNWs vasculogenic gold nanowires
- FIG. 1B illustrates hiPSC-CMs functionalized with multipurpose GNWs, in accordance with one or more embodiments.
- FIG. 1C illustrates scaffold-free micro-tissue with architectural arrangement of hiPSC-CMs and cardiac fibroblasts (CFs), in accordance with one or more embodiments.
- FIG. 1D illustrates cell-cell coupling and engraftment with host tissue, in accordance with one or more embodiments.
- FIG. 1E illustrates enhanced electromechanical integration (i.e. synchronous contraction) of the micro-tissues with the host myocardium due to high conductivity properties of GNWs.
- FIG. 1F illustrates neovascular formation within the micro-tissues due to conjugation of VEGF-mimetic vasculogenic peptide to GNWs, in accordance with one or more embodiments.
- FIG. 2A illustrates synthesized GNRs incorporated within gelatin-based hydrogels (GelMA-GNRs) with improved electrical, structural properties, in accordance with one or more embodiments.
- FIG. 2B illustrates the electrical properties of GNRs incorporated within gelatin-based hydrogels (GelMA-GNRs), in accordance with one or more embodiments.
- FIG. 2C illustrates GNR concentration v. viability of neonatal rat CMs seeded on GelMA-GNWs tissues, in accordance with one or more embodiments.
- FIG. 2D illustrates homogeneous distribution of cardiac specific markers (sarcomeric ⁇ -actinin) and Connexin43 (CX43) gap junction protein along with increased cell binding to the matrix (integrin ⁇ -1) confirmed the formation of an integrated tissue layer, in accordance with one or more embodiments.
- FIG. 2E illustrates conductive GNRs embedded gelatin scaffolds significantly supported synchronous tissue-level contractility and calcium transient of CMs, in accordance with one or more embodiments.
- FIG. 3 illustrates a reaction synthetic scheme of GNW-PEGPeptide from GNW-CTAB via EDC/Sulfo NHS chemistry, in accordance with one or more embodiments.
- FIG. 4 illustrates a generation of scaffold-free micro-tissues comprised of co-culture of GNWs functionalized hiPSCs-CMs and CFs, in accordance with one or more embodiments.
- FIG. 5A illustrates a schematic diagram of the process of developing and implementing the GNW-RGD cardiac spheroid, in accordance with one or more embodiments.
- FIG. 5B illustrates a TEM micrograph of GNW-CTAB, in accordance with one or more embodiments.
- FIG. 5C illustrates a TEM micrograph of GNW-RGD, in accordance with one or more embodiments.
- FIG. 5D illustrates viability results of GNW-CTAB, in accordance with one or more embodiments.
- FIG. 5E illustrates viability results of GNW-RGD, in accordance with one or more embodiments.
- FIG. 5F illustrates normalized metabolic activity rate of cardiomyocytes, in accordance with one or more embodiments.
- FIG. 5G illustrates a normalized number of nuclei per mm 2 showing cell retention on day of culture, in accordance with one or more embodiments.
- FIG. 6 illustrates a schematic diagram of decorated gold nanostructures and the preparation thereof, in accordance with one or more embodiments.
- Gold Nanostructures and microengineering technology are used to develop an injectable electrically conductive spheroid-based micro-tissue, embedded with gold nanowires or nanorods, for functional regeneration of infarcted myocardium.
- the developed micro-tissues can be injected (or implanted) within the infarcted region of myocardium to restore the tissue function loss and prevent heart failure (See FIG. 5A-5G ).
- the electrically conductive cardiac micro-tissues provide a desirable microenvironment to enhance the functionalities of cardiac cells and to better integrate to the native heart.
- Biocompatible and functionalized gold nanowires are synthesized to decorate the intercellular microenvironment of the cardiac microtissues.
- the GNWs were capped with polyethylene glycol bi-linker (COOH-PEG-SH) and functionalized using RGD cell adhesion motifs to 1) increase the biocompatibility of the conventional GNWs capped with toxic surfactant (Cetrimonium bromide, CTAB), 2) induce high negative surface charge to decrease the intracellular uptake, and 3) increase the GNW-cell interaction to enhance the spheroid structural integrity.
- COOH-PEG-SH polyethylene glycol bi-linker
- CTAB toxic surfactant
- Spheroid-based cardiac micro-tissues with well defined geometrical features and mix functionalized GNWs with the micro-tissues are used to develop the final electrically conductive and injectable spheroid based cellular clusters.
- the procedure is minimally invasive, given that our injectable and electrically conductive micro-tissues can be directly delivered via catheter to the infarcted zone of myocardium (i.e., chest cavity not opened).
- the micro-tissues can be delivered via a minimally invasive surgical catheter based delivery.
- the developed tissues could be used for in vivo testing in both small and large animal models.
- the preparation and functionalization of the GNWs can be conducted in a number of different manners.
- preparation and functionalization of GNWs: and GNW-CTAB with average aspect ratio of 21 was synthesized.
- GNWs capped with SH-PEG-COOH was synthesized via a customized 2-step exchange procedure including primary PEGylation in Tris buffer (pH 3, 24 hr) followed by post-PEGylation in ethanol (20%, 24 hr).
- RGD functionalized GNWs (GNW-RGD) was synthesized using GNW-COOH and RGD peptide based on EDC/NHS protocol. Transmission electron microscopy, Raman spectroscopy and dynamic light scattering were used to characterize the produced GNWs (-CTAB, —COOH and -RGD).
- PDMS polydimethylsiloxane
- Agarose concave microwells are used based on well-established protocols and our preliminary studies to engineer micro-tissues. Briefly, a micro-engineered master composed of an array of 20 ⁇ 20 concave microwells in the range of 100-300 ⁇ m diameter and 150 ⁇ m in spacing will be purchased or fabricated using SU-8 photoresist and soft lithography technique. Subsequently, a 10:1 mixture of elastomer and curing agent will be poured on PDMS master and racked out using a glass slide by applying light pressure. Surface tension will allow formation of meniscus on cylindrical microwells of PDMS master leading to formation of concave microwells.
- Cardiac cells mixed with functionalized GNWs having concentrations of 1, 10 and 50 micro-grams/ml will be seeded on microwells at a density of 10-15 ⁇ 10 ⁇ circumflex over ( ) ⁇ 6 cells/ml of cardiac media. Passive cell seeding is expected to lead to formation of cellular clusters within each microwell. Upon cell seeding, microwells will be placed inside 37° C. for 7 days to form micro-tissues.
- a unique and integrated strategy is proposed to develop next generation of MRT based on nanoengineering of scaffold-free cardiac microtissues using electrically conductive, cell adhesion-promoting and vasculogenic gold nanowires (GNWs) ( FIG. 1A ).
- CMs cardiovascular cells
- FIG. 1B GNWs
- Microscale technologies will be used to generate the scaffold-free tissues comprised of co-culture of functionalized CMs and cardiac fibroblast (CFs), with specific architectural arrangement ( FIG. 1C ).
- the architectural arrangement may include a plurality of CFs arranged in at least one layer of fibroblasts that surrounds, encompasses, encircles, envelops, or encapsulates a cluster of CMs (e.g., functionalized CMs).
- CMs e.g., functionalized CMs
- other architectural arrangements may be utilized. Since, gold nanomaterials-embedded scaffolds improve functional outcome in MRT, direct functionalization of CMs with multipurpose GNWs will allow scaffold-free MRT constructs that will lead to: a) establishment of mature cell-cell coupling due to GNWs that are conjugated with adhesion-promoting RGD peptide ( FIG. 1D ); b) enhanced electromechanical integration (i.e.
- FIG. 1E synchronous contraction of the micro-tissues with the host myocardium due to high conductivity properties of GNWs
- FIG. 1F promotion of neovascular formation within and from the host towards the micro-tissues due to conjugation of VEGF-mimetic peptide to GNWs
- Addition of CFs, with specific architectural arrangement i.e. within the outer layer of microtissues, will further enhance engraftment of the micro-tissues with the host while simultaneously promoting a native like endogenous niche through ECM production and paracrine CM-CF signaling.
- the proposed strategy produces injectable micro-tissues for intramyocardial delivery via minimally invasive catheter-based approach.
- scaffold-free, electrically conductive and vasculogenic cardiac micro-tissue include hiPSCs-CMs and CFs.
- in vitro maturity and functionalities of micro-tissues including GNWs conjugated with RGD and VEGF-mimetic QK peptides is assessed.
- scaffold-free cardiac micro-tissues including GNW-functionalized hiPSCs-CMs and CFs are generated for in vitro biological assessments.
- micro-tissues will work in an in vivo model, such as in preclinical rodent model and eventually in clinical applications. This will improve myocardial function, vascular formation and integration of micro-tissues within the host myocardium.
- the utility of the injected micro-tissues in improving myocardial function can be evaluated, and vascular formation and integration of the micro-tissues with the host myocardium are investigated.
- CMs with multipurpose GNWs functionalization of the surface of CMs with multipurpose GNWs are conjugated with cell adhesion promoting and vasculogenic peptides.
- Native ventricular myocardium consists of electrically conductive Purkinje fibers coupled with tightly packed cellular constructs consisting mainly of CMs and CFs.
- conductive nanomaterials i.e. carbon nanotubes (CNTs), graphene oxide (GO), silicon (SO) nanowires, gold nanostructure
- Nanoengineering is used to functionalize for the first time the surface of human induced pluripotent stem cell derived CMs (hiPSC-CMs), which will form the primary building blocks of the micro-tissues.
- hiPSC-CMs human induced pluripotent stem cell derived CMs
- GNWs are used that are intrinsically electrically conductive, but in addition, the surface of GNWs are conjugated with cell adhesion-promoting RGD peptide ( FIG. 1A ).
- Neovascularization Our approach is also designed to enhance neovascular formation by conjugation of vasculogenic VEGF-mimetic peptide (along with RGD), on the surface of GNWs. This strategy will address a significant limitation of current MRT approaches that lack vascular development to support and sustain implanted or injected engineered tissue constructs.
- Scaffold-free cardiac micro-tissues with specific architectural arrangement In our unique strategy, microscale technology (i.e. use of thermo-responsive microwells) is used to develop scaffold-free cardiac micro-tissues comprised of co-culture of functionalized hiPSC-CMs and CFs with specific architectural arrangement.
- CFs are arranged within the outer layer of the micro-tissues with the primary purpose of promoting the engraftment of the micro-tissues with the host upon injection through cell-ECM interaction and production of native ECM proteins (i.e. collagen) by CFs.
- Our approach is the first strategy to generate cardiac micro-tissues with this unique architectural arrangement.
- Native myocardium is a multi-cellular and adaptive tissue consisting of myocytes and non-myocyte cells.
- the embodiments herein develop scaffold-free, electrically conductive and vasculogenic cardiac micro-tissues comprised of hiPSCs-CMs and CFs.
- Efficient MRT of infarcted myocardium relies on critical factors including proper cell source, enhanced retention and cell-cell coupling and engraftment, electromechanical integration of the engineered tissues with the surrounding host myocardium and neovascularization.
- GNWs conjugated with RGD and VEGF-mimetic QK peptides GNWs for example, with the average aspect ratio of 24 ( ⁇ 5 ⁇ m in length and ⁇ 50 nm in diameter) will be synthesized based on seed-mediated anisotropic growth method.
- CTAB-capped GNWs will be centrifuged (2000 rpm, 20 min) two times and re-dispersed in 1 mM CTAB solution to reduce the concentration of CTAB to the critical micelle formation concentration (CMC in water for CTAB is ⁇ 1 mM).
- the supernatant will be discarded, and 400 ⁇ L of Tris buffer (50 mM, pH 3) will be added drop-wise to the GNWs pellet.
- Tris buffer 50 mM, pH 3
- 30 ⁇ L of COOH-PEG-SH (2 mM in DIW) will be added to the GNW-Tris mixture under vortexing and kept agitated for 1 min.
- the final mixture will be maintained undisturbed for 24 hr at room temperature to allow the completion of the PEGylation.
- the mixture will be then centrifuged (4000 rpm, 25 min) to remove the unreacted PEG bi-linkers, Tris buffer and free CTAB molecules.
- the freshly synthesized GNWPEG-COOH will be gently re-dispersed in 20% ethanol, followed by addition of 30 ⁇ L of COOH-PEG-SH (2 mM in 20% ethanol) to the mixture under gentle vortexing. The mixture will be kept undisturbed at room temperature for 24 hr.
- the two-step functionalized GNW-PEG-COOH will be harvested by centrifugation at 4000 rpm (25 min), and re-dispersed in 500 ⁇ L PBS.
- the carboxylic acid groups of GNW-PEG-COOH will be activated by adding EDC and Sulfo-NHS forming the corresponding GNW-PEG-COO—NHS ester.
- EDC electrospray diluent
- Sulfo-NHS adenosine triphosphate
- GNW-PEGCOOH dispersed in 500 ⁇ L PBS (obtained from the previous step) will be added with 2 mg of EDC, vortex for 20 sec and then 5.5 mg of Sulfo-NHS will be added to this solution at room temperature for 10 min.
- GNW-PEG-RGD and GNW-PEG-QK mixture will be purified by centrifugation/decantation in PBS for 1 time.
- subsequent characterization will be performed using TEM, FTIR, Raman spectra and NMR. The presence and the size of GNWs will be obtained from TEM images.
- the conjugation of Au to the —SH group of HS-PEG-COOH will be characterized by the appearance of Au—S shift in Raman spectra. Furthermore, GNWs of the GNW-PEG-Peptides will be digested prior to taking NMR spectra. The appearance of amide I, amide II and amide A bands in the FTIR spectra and the appearance of the chemical shift corresponding to N—H bond in NMR spectra will confirm the presence and amount of peptides conjugated to GNWs.
- One or more embodiments herein will generate scaffold-free cardiac micro-tissues, using GNW-functionalized hiPSCs-CMs and CFs, and generate micro-tissues comprised of hiPSCs-CMs and CFs.
- CFs will be transfected to stably express yellow fluorescent proteins (CF-YFP) for tracking within the micro-tissues, while enabling 3-color imaging.
- An initial co-culture ratio of 3:1 (hiPSCs-CMs:CFs) will be selected based on our previous studies to enhance tissue-level function.
- PNIPAAam-based microwells (circular, starting with 250-300 ⁇ m diameter, 300 ⁇ m depth) along with twostep cell seeding process (hiPSCs-CMs followed by CFs, 3:1 ratio) ( FIG. 3 ).
- hiPSCs-CMs (10 ⁇ 106 cells/ml in culture media) will be mixed with different concentrations of functionalized GNWs (0, 2.5 and 5 ⁇ g/ml) and will be seeded on the microwells at 25° C.
- the microwells will be rinsed in PBS to remove excess cells.
- microwells will be placed inside 37° C. for 2 h to increase surface area and volume, opening up room for seeding CFs ( FIG. 4 ).
- microwells Upon sequential seeding of both cell types, microwells will be placed inside 37° C. for 7 days for the formation of final micro-tissues.
- One or more embodiments include visualization of the GNWs on cell surface. For example, upon 1, 2, and 3 weeks, tissue sectioning and TEM imaging are utilized to investigate the localization of GNWs on cell membrane within the micro-tissues. Briefly, microtissues are fixed in 2.5% glutaraldehyde and post-fixed in osmium tetroxide (1%) with 1.5% K+ ferricyanide. Fixed samples will then be dehydrated in ethanol/propylene oxide and embedded in epoxy resin for sectioning. Ultrathin slices (100 nm, Ultra microtome) are prepared and stained using uranyl acetate and lead citrate.
- assessment of cell-cell coupling within the micro-tissues is done as follows. On 1, 2, and 3 weeks of culture, the micro-tissues are fixed in 4% (v/v) paraformaldehyde (PF) in PBS and immunohistochemistry (IHC) consistent with our previous studies, will then be performed in each experimental condition (mono- and co-culture with defined concentrations of GNWs) to co-stain CX40, CX43 and CX45 gap junction proteins (Alexa Fluor 595, different samples) with sarcomeric proteins (MLC2a, MLC2v, ⁇ -actinin, ⁇ -MHC, Alexa Fluor 488). Within all the samples, the cells' nuclei will be stained with DAPI.
- PF paraformaldehyde
- IHC immunohistochemistry
- beating frequency beats per min, BPM
- Side-by-side of contractility analysis we will utilize IHC images to analyze for sarcomere organization within the selected regions of interest (ROIs, 300 ⁇ 300 ⁇ m2) within each condition. Consistent to our previous work, this analysis will determine whether conjugated GNWs impart a significant enhancement on contractility of the micro-tissues correlated to sarcomere organization.
- Enhanced contractility of the micro-tissues i.e. spontaneous beating synchrony
- Intracellular calcium (Ca2+) transients and electrophysiologic responsiveness of the micro-tissues For Ca2+ transients measurements, samples will be exposed to 2.3 mM fluo-4 AM and 0.1% Pluronic F-127 for 15 min at 37° C. After 15 min, samples will be washed 3 ⁇ in Tyrode's solution. Ca2+ transients will be imaged using confocal microscopy (63 ⁇ ) at 5-10 locations of each sample. Additionally, we will utilize qPCR analysis to assess upregulation of genes handling calcium proteins (i.e. SERCA2, RYR2, CACNA1C) in presence of GNWs. Within different samples, conduction velocity of cardiac micro-tissues will be analyzed using voltage sensitive dye RH-237. To determine contractile responsiveness to extraneous electrophysiologic signal (to mimic electrical signal from host myocardium), micro-tissues will be stimulated using platinum electrodes with 0.5-2 Hz, 5 V and 1-2 ms biphasic square pulses.
- action potential (AP) will be measured (5-10 locations) and conduction velocity quantified.
- Expected outcomes Synchronized calcium (Ca2+) transients spikes across multiple ROIs of the microtissues as well as increased upregulation of genes handling calcium proteins as function in groups conjugated with GNWs. Increased level of action potential in co-culture groups with conjugated GNWs.
- vascular formation We utilized a microfluidic chip to assess utility of VEGF-mimetic QK peptide in promoting vascularization within 3D gelatin-based hydrogels.
- human umbilical vein endothelial cells (HUVECs, density: 15 ⁇ 106 cells/ml) were encapsulated within gelatin based hydrogel matrix (thiolated gelatin, Gel-S) conjugated with variable concentrations of QK mathacrylate peptide (sequence: KLTWQELYQLKYKGI-C (SEQ ID NO: 2), 0, 100, 150, 200 ⁇ g/mL).
- QK mathacrylate peptide sequence: KLTWQELYQLKYKGI-C (SEQ ID NO: 2), 0, 100, 150, 200 ⁇ g/mL.
- incorporation of QK peptide resulted in formation of robust, inter-connected vascular network with significantly increased average branch length, branch diameter and vascular network coverage.
- We will utilize a similar assay to encapsulate HUVECs along with micro-tissues (scaffold-free) within the microfluidic platform to assess the role of VEGF mimetic QK peptide, conjugated on GNWs, on vascular formation within the surrounding of the micro-tissues.
- GNWs if cellular uptake of GNWs occurs, we will adjust our synthesis process to increase the length to 7-10 ⁇ m. In case of cellular contraction and decrease in the size of micro-tissues to 150 ⁇ m, we will increase the diameter of the microwells ( ⁇ 300-350 ⁇ m). In case, if we do not observe statistically significant difference in contractility or Ca2+ transients in presence of GNWs, we will optimize and increase the concentration in the range of 7.5 to 10 ⁇ g/ml.
- Embodiments further optionally include evaluating the functionalities of the developed cardiac micro-tissues in vivo in a rodent model (Months 16-24).
- Injectable cardiac micro-tissues developed herein, are envisioned to significantly promote MRT through enhanced cell-cell coupling, cell-matrix interaction, electrical engraftment with the host myocardium and nonvascular formation. Therefore, it is important to evaluate biological and physiological performance of the injectable micro-tissue in vivo. In this, we will use small animal model (rodent) to study whether the developed cardiac micro-tissues will lead to such improved biological and physiological functionalities.
- MRT next generation of MRT is discussed herein, utilizing a multidisciplinary and state-of-the-art approach based on nanoengineered hiPSCs-CMs functionalized with multipurpose GNWs.
- the embodiments provide for the ultimate development of injectable micro-tissues with superior cell-cell coupling, engraftment, electromechanical integration and neovascularization leading to effective functional MTR.
- the gold nanostructures can be selected to optimize the utility of the resulting microtissue. Accordingly, the nature of the gold nanostructures is not critical, provided the resulting scaffold-free micro tissue possesses the desired physical properties and biological function.
- the gold nanostructures can be 1D structures such as a wires, rods, or spheres.
- the gold nanowires will have a width of about 20 nm to about 50 nm and a length of up to about 5 ⁇ m.
- Gold nanorods will typically have a width of about 50 nm and a length of up to about 100 nm, about 200 nm or about 300 nm.
- Gold nanospheres will typically have a diameter of up to about 50 nm or up to about 100 nm.
- the gold nanostructures can also include 2D structures such as nano-plates.
- the gold nanoplates will be 2D sheets having dimensions up to about 250-500 nm ⁇ 250-500 nm.
- the gold nanostructures 100 can be linked to the cell-adhesion moiety or to the functional moiety through a direct chemical bond or through a linking group X, as shown in FIG. 6 .
- the gold nanostructure is linked to the cell-adhesion moiety or to the functional moiety through a direct chemical bond.
- the gold nanostructure is linked to the cell-adhesion moiety or to the functional moiety through a linking group X. Any linking group that provides a GNW with desired properties and function may be used.
- the linker has a molecular weight of from about 20 daltons to about 20,000 daltons.
- the linker has a molecular weight of from about 20 daltons to about 5,000 daltons.
- the linker has a molecular weight of from about 20 daltons to about 1,000 daltons.
- the linker has a molecular weight of from about 20 daltons to about 200 daltons.
- the linker has a length of about 5 angstroms to about 60 angstroms.
- the linker separates the antigen from the remainder of the compound of formula I by about 5 angstroms to about 40 angstroms, inclusive, in length.
- the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (—O—), and wherein the chain is optionally substituted on carbon with one or more (e.g.
- the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (—O—), and wherein the chain is optionally substituted on carbon with one or more (e.g.
- the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C 1 -C 6 )alkoxy, (C 3 -C 6 )cycloalkyl, (C 1 -C 6 )alkanoyl, (C 1 -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, (C 1 -C 6 )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo ( ⁇ O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- substituents selected from (C 1 -C 6 )alkoxy, (C 3 -C 6 )cycloalkyl, (C 1 -C 6 )alkanoy
- the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C 1 -C 6 )alkoxy, (C 3 -C 6 )cycloalkyl, (C 1 -C 6 )alkanoyl, (C 1 -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, (C 1 -C 6 )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo ( ⁇ O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- substituents selected from (C 1 -C 6 )alkoxy, (C 3 -C 6 )cycloalkyl, (C 1 -C 6 )alkanoy
- the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms.
- the linker is a divalent, branched or unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.
- the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.
- the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 6 carbon atoms.
- the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 4 carbon atoms.
- the linker comprises a polyethyleneoxy chain.
- the polyethyleneoxy chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating ethyleneoxy units.
- the linker is a divalent radical formed from a peptide.
- the linker is a divalent radical formed from an amino acid.
- the linker is a divalent radical of formula —S-(PEG)-C( ⁇ O)—, as illustrated in FIG. 3 , wherein C( ⁇ O)— is bonded to the amino terminus of the RGD peptide.
- the molecular weight of PEG is 3500 Dalton g/mol.
- amino acid comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g.
- the term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g.
- acetyl or benzyloxycarbonyl as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C 1 -C 6 )alkyl, phenyl or benzyl ester or amide; or as an ⁇ -methylbenzyl amide).
- suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Synthesis ; Wiley: New York, 1981, and references cited therein).
- An amino acid can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine.
- peptide describes a sequence of 2 to 25 amino acids (e.g. as defined hereinabove) or peptidyl residues.
- the sequence may be linear or cyclic.
- a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence.
- a peptide can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine.
- a peptide comprises 3 to 25, or 5 to 21 amino acids.
- Peptide derivatives can be prepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, or as described in the Examples hereinbelow. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.
- the gold nanostructures can be linked to one or more cell adhesion moieties.
- the cell adhesion moiety provides affinity for targeting of the gold nanostructures toward the cell membrane. Additionally, these cell adhesion moieties are envisioned to enhanced cell-cell coupling among cardiomyocytes as well as cell cell-matrix interaction and overall cellular retention between the micro-tissues and the surrounding host matrix upon implantation.
- the cell adhesion moiety would be a peptide, which is conjugated to the surface of the gold nanostructures.
- the cell adhesion moiety is an integrin binding peptides such as RGD or DGEA (SEQ ID NO: 3) or other peptides GRGDSP (SEQ ID NO: 4) or GRGDY (SEQ ID NO: 5) peptides.
- peptides belong to classes of synthetic peptides that contain the amino acids: specifically for RGD: Arg-Gly-Asp, for DGEA (Asp-Gly-Glu-Ala) (SEQ ID NO: 3), for GRGDSP (H-Gly-Arg-Gly-Asp-Ser-Pro-OH) (SEQ ID NO: 4) and for GRGDY (Gly-Arg-Gly-Asp-Tyr) (SEQ ID NO: 5).
- RGD Arg-Gly-Asp
- DGEA Asp-Gly-Glu-Ala
- GRGDSP H-Gly-Arg-Gly-Asp-Ser-Pro-OH
- GRGDY Gly-Arg-Gly-Asp-Tyr
- the gold nanostructures can be linked to one or more functional moieties.
- the functional moiety provides for specific target functionalities such as promoting vascular formation toward the injected tissues, reduce inflammatory response upon injection of the cells or reduce apoptosis.
- the functional moiety is a peptide.
- the function moiety could also be aptamers (oligonucleotides).
- the functional moiety is a vasculogenic peptide, an anti-inflammatory peptide, antiapoptotic/antinecrotic peptides.
- the functional units and/or moieties includes one or more of vascularization peptides, anti-inflammatory peptides, antiapoptotic/antinecrotic peptides, or antioxidant peptides.
- vascularization peptides include one or more of vascularization peptides, anti-inflammatory peptides, antiapoptotic/antinecrotic peptides, or antioxidant peptides.
- vasculogenic peptides we can have: 1-QK (Methacrylic acid-K(Ac)LTWQELYQLK(Ac)YK(Ac)GI-NH2 (SEQ ID NO: 1)).
- VEGF memetic peptide [SLanc: K-(SL)3(RG)(SL)3-K-G-KLTWQE-LYQLKYKGI (SEQ ID NO: 6)].
- the preparation of certain gold nanostructures that can be incorporated into the microtissues of the invention is illustrated in FIG. 6 .
- the gold nanostructure can be linked to a linking group (X), which can then be modified to incorporate an activating group (Y) that can be used to facilitate the attachment of the cell adhesion moiety or the functional moiety.
- the gold nanostructure can be linked directly to a group —X-Y in one step.
- Processes and reagents that can be used to modify a gold surface are known, for example, see Zhang, Z. & Lin, M. Fast loading of PEG-SH on CTAB-protected gold nanorods. RSC Adv. 4, 17760-17767 (2014), and/or Kinnear, C. et al. Gold Nanorods: Controlling Their Surface Chemistry and Complete Detoxification by a Two-Step Place Exchange. Angew. Chem. Int. Ed. 52, 1934-1938 (2013).
- the linking group X can be attached to the gold nanostructure in any synthetically feasible linkage.
- the gold surface can be attached to the linking group X through a covalent bond with a sulfur atom of X.
- the activating group Y can be a leaving group that can be displaced by an atom on the cell adhesion moiety or on the functional moiety—or the activating group Y can be a reactive group that can react with an atom on the cell adhesion moiety or on the functional moiety.
- the activating group is sulfo N-hydroxysuccinamide (as illustrated in FIG. 3 ) or another activated group that is capable of forming an amide with an amine group of the cell adhesion moiety or on the functional moiety. Such activated amide forming groups and reaction conditions are known.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Transplantation (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Dermatology (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Inorganic Chemistry (AREA)
- Developmental Biology & Embryology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
- This application is a divisional of U.S. patent application Ser. No. 16/157,956, filed Oct. 11, 2018, which claims priority to U.S. Provisional Application Ser. No. 62/571,056, filed on Oct. 11, 2017. The entire content of the applications referenced above are hereby incorporated by reference herein.
- The instant application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 29, 2019, is named 17555_057US1_SL.txt and is 2,203 bytes in size.
- Approximately 6.5 million people in the U.S. are diagnosed with heart failure, with 4134 patients (May 2016) on a waiting list to receive a heart transplant. Cardiovascular diseases, including myocardial infarction (MI), remain a leading cause of mortality and morbidity worldwide, accounting for over 40% of all human death. MI leads to loss of cardiomyocytes (CMs), which have limited self-regenerative capacity, leading to impaired contractility, abnormal stress distribution throughout the heart, adverse global remodeling and ultimately heart failure. Heart transplantation or implantation of mechanical left ventricular assist device are treatment options of last resort, but are limited by inadequate organ donors and potential complications of the surgical procedures. For the vast majority of heart failure patients, treatment is limited to pharmacologic therapy to optimize the function of remaining CMs and slow the adverse cardiac remodeling. Those skilled in the art will appreciate that cardiomyocytes and cardiac myocytes are different terms for the same types of cells.
- Over the last decade, alternative approaches were developed for myocardial replacement therapy (MRT), but results have so far been disappointing. These strategies comprised of cell-based transplantation, injectable biomaterials (i.e. cell-laden or acellular) or engineered tissue constructs. The common goal of these approaches is to restore native-like functionalities (i.e. enhanced contractility) while maintaining structural integrity of the myocardium.
- The clinical use of cell-based MRT has been hampered by the high rate of cell loss and poor cell-cell coupling and engraftment with host myocardium. Cell-based MRT involves local injection of stem/progenitor or adult cells into infarcted myocardium to augment existing pool of CMs and initiate an endogenous regeneration process. This approach has many potential advantages including use of less invasive procedures, lack of reliance on availability of donor hearts and use of patient's own cells to obviate immune reactions. Cell-based MRT approaches include of direct injection of adult cells (e.g. skeletal myoblasts, mesenchymal), cardiac stem/progenitor cells (e.g. C-kit27 cardiac side population, sca, Islet, cardiosphere) as well as human pluripotent stem cells (hiPSCs). Current cell-based MRT approaches, however, are hampered by poor (˜50%) cell retention, inadequate cell-cell coupling and poor engraftment with the host myocardium, with only 10% survival of injected cells. The shortcomings of cell-based MRT generated interest in embedding the desired cells into scaffolding matrices to serve as “tissue engineered cardiac patches” or “injectable cell-laden biomaterials” (i.e. scaffold-based approaches) that could be locally applied to the injured zone. Various approaches utilized natural (e.g. alginate, fibrin, collagen, extracellular matrices) or synthetic materials (i.e. PNIPAAm, self-assembling peptides) for injection or implantation (i.e. patch) of cells into the infarcted region. However, in vivo pre-clinical studies of cell-embedded biomaterials have so far demonstrated inconsistent outcomes due to lack of electromechanical integration (i.e. synchronous contraction) of the tissues with the host myocardium, raising the potential risk of arrhythmias, as well as poor vascularization. Furthermore, implantation of cell-embedded patches can potentially cause immune rejection or foreign body reactions while requiring a highly invasive approach (i.e. open-heart surgery) to implant the tissues.
- Regenerative Medicine and tissue engineering strategies offer promising avenues to address the current limitations in organ transplantation in general, and cardiac repair in particular. Native ventricular myocardium consists of CMs coupled with electrically conductive purkinje fibers and mechanically robust extracellular matrix (ECM). This unique architecture exhibits tightly packed and aligned (i.e. anisotropic) cellular constructs. Following injury, replacement of infarcted myocardium would require proper scaffolding biomaterials and cell sources to mimic the structural architecture of native myocardium. Although substantial progress has been made in the synthesis of new biomaterials to replace injured cardiac tissue using natural or synthetic polymers (collagen, elastin, silk, gelatin, etc), unfortunately, the electrical properties of these hydrogels do not come close to the properties of the native myocardium. Inadequate cell adhesion sites and electrically insulated structure of conventional hydrogels lead to poor tissue-level functionalities, lack of integration with the host myocardium, and ultimately failure of the tissue engineered constructs.
- To date, several studies have demonstrated that employing electrically conductive nanomaterials enables addressing the shortcomings of conventional hydrogel-based scaffolds with respect to their electrical conductivity. Carbon nanotubes (CNT) have been among well respected conductive nanomaterials for cardiac tissue engineering. CNTs-embedded scaffolds have particularly demonstrated enhanced electrical properties that facilitated electrical signal propagation and cell-cell coupling. While incorporation of CNTs results in superior properties, several controversial cytotoxicity issues have raised numerous concerns for their use in clinical applications.
- Due to these critical shortcomings, there is still an unmet need to develop treatment strategies for long-term regeneration of injured myocardium.
- A scaffold-free microtissue comprising gold nanostructures within the microtissue.
- In one or more embodiments, the microtissue comprises cardiac myocytes, endothelial cells, pluripotent stem cells, myoblasts, or fibroblasts.
- In one or more embodiments, the gold nanostructures are 1D gold nanostructures.
- In one or more embodiments, the 1D gold nanostructures are wires, rods, or spheres.
- In one or more embodiments, the gold nanostructures include 2D gold nanostructures.
- In one or more embodiments, the 2D gold nanostructures are nano-plates.
- In one or more embodiments, one or more gold nanostructures are linked to a cell adhesion moiety.
- In one or more embodiments, the cell adhesion moiety is a peptide.
- A cardiac microtissue comprising nanoscale decoration of cardiac cells including gold nanostructures within the microtissue.
- In one or more embodiments, the microtissue is scaffold free.
- In one or more embodiments, the gold nanostructures are functionalized using RGD cell adhesion motifs.
- In one or more embodiments, the gold nanostructures are capped with polyethylene glycol bi-linker.
- In one or more embodiments, the microtissue is spheroid based.
- In one or more embodiments, the gold nanostructures are conjugated with vasculogenic peptides.
- In one or more embodiments, the microtissue further includes functionalized cardiomyocytes and cardiac fibroblasts within the gold nanostructures.
- In one or more embodiments, an electrically conductive composition comprising cardiac micro-tissue and gold nanostructures is described herein.
- In one or more embodiments, a method for regeneration or repair of infarcted myocardium in an animal comprises injecting a composition as described above.
- In one or more embodiments, the composition is injected within the infarcted region of myocardium of the animal.
- In one or more embodiments, an electrically conductive composition as described above for use in medical therapy.
- In one or more embodiments, an electrically conductive composition as described above for regeneration or repair of infarcted myocardium.
- In one or more embodiments, the use of an electrically conductive composition as described above to prepare a medicament for regeneration or repair of infarcted myocardium.
- Consistent with the disclosed embodiments, a scaffold-free microtissue is disclosed. The scaffold-free microtissue comprises one or more gold nanostructures linked to a functional moiety, wherein the functional moiety is one or more vasculogenic peptides, one or more anti-inflammatory peptides, one or more antiapoptotic peptides, one or more antinecrotic peptides, one or more antioxidant peptides, one or more oligonucleotides, one or more lipid particles, one or more phospholipid particles, one or more liposomes, one or more nanoliposomes, one or more microRNAs, or one or more siRNAs. The scaffold-free microtissue further comprises a plurality of cardiac myocytes or cardiac myoblasts, wherein the cardiac myocytes or cardiac myoblasts are conjugated to the one or more gold nanostructures, wherein the plurality of cardiac myocytes or cardiac myoblasts are arranged in a cluster. The scaffold-free microtissue further comprises a plurality of fibroblasts, wherein the fibroblasts are arranged in at least one layer of fibroblasts that substantially surrounds the cluster of gold-nanostructure-conjugated cardiac myocytes or gold-nanostructure-conjugated cardiac myoblasts.
- Consistent with the disclosed embodiments, a method for regeneration or repair of an infarcted myocardium including an infarcted region in an animal comprises injecting a scaffold-free microtissue into the animal.
-
FIG. 1A illustrates conjugated, scaffold-free cardiac micro tissues using electrically conductive, cell adhesion-promoting and vasculogenic gold nanowires (GNWs), in accordance with one or more embodiments. -
FIG. 1B illustrates hiPSC-CMs functionalized with multipurpose GNWs, in accordance with one or more embodiments. -
FIG. 1C illustrates scaffold-free micro-tissue with architectural arrangement of hiPSC-CMs and cardiac fibroblasts (CFs), in accordance with one or more embodiments. -
FIG. 1D illustrates cell-cell coupling and engraftment with host tissue, in accordance with one or more embodiments. -
FIG. 1E illustrates enhanced electromechanical integration (i.e. synchronous contraction) of the micro-tissues with the host myocardium due to high conductivity properties of GNWs. -
FIG. 1F illustrates neovascular formation within the micro-tissues due to conjugation of VEGF-mimetic vasculogenic peptide to GNWs, in accordance with one or more embodiments. -
FIG. 2A illustrates synthesized GNRs incorporated within gelatin-based hydrogels (GelMA-GNRs) with improved electrical, structural properties, in accordance with one or more embodiments. -
FIG. 2B illustrates the electrical properties of GNRs incorporated within gelatin-based hydrogels (GelMA-GNRs), in accordance with one or more embodiments. -
FIG. 2C illustrates GNR concentration v. viability of neonatal rat CMs seeded on GelMA-GNWs tissues, in accordance with one or more embodiments. -
FIG. 2D illustrates homogeneous distribution of cardiac specific markers (sarcomeric α-actinin) and Connexin43 (CX43) gap junction protein along with increased cell binding to the matrix (integrin β-1) confirmed the formation of an integrated tissue layer, in accordance with one or more embodiments. -
FIG. 2E illustrates conductive GNRs embedded gelatin scaffolds significantly supported synchronous tissue-level contractility and calcium transient of CMs, in accordance with one or more embodiments. -
FIG. 3 illustrates a reaction synthetic scheme of GNW-PEGPeptide from GNW-CTAB via EDC/Sulfo NHS chemistry, in accordance with one or more embodiments. -
FIG. 4 illustrates a generation of scaffold-free micro-tissues comprised of co-culture of GNWs functionalized hiPSCs-CMs and CFs, in accordance with one or more embodiments. -
FIG. 5A illustrates a schematic diagram of the process of developing and implementing the GNW-RGD cardiac spheroid, in accordance with one or more embodiments. -
FIG. 5B illustrates a TEM micrograph of GNW-CTAB, in accordance with one or more embodiments. -
FIG. 5C illustrates a TEM micrograph of GNW-RGD, in accordance with one or more embodiments. -
FIG. 5D illustrates viability results of GNW-CTAB, in accordance with one or more embodiments. -
FIG. 5E illustrates viability results of GNW-RGD, in accordance with one or more embodiments. -
FIG. 5F illustrates normalized metabolic activity rate of cardiomyocytes, in accordance with one or more embodiments. -
FIG. 5G illustrates a normalized number of nuclei per mm2 showing cell retention on day of culture, in accordance with one or more embodiments. -
FIG. 6 illustrates a schematic diagram of decorated gold nanostructures and the preparation thereof, in accordance with one or more embodiments. - In one or more embodiments, Gold Nanostructures and microengineering technology are used to develop an injectable electrically conductive spheroid-based micro-tissue, embedded with gold nanowires or nanorods, for functional regeneration of infarcted myocardium.
- Electrically conductive and highly functional cardiac micro-tissues nano-engineered with gold nanowires for regeneration and repair of infarcted myocardium. The developed micro-tissues can be injected (or implanted) within the infarcted region of myocardium to restore the tissue function loss and prevent heart failure (See
FIG. 5A-5G ). The electrically conductive cardiac micro-tissues provide a desirable microenvironment to enhance the functionalities of cardiac cells and to better integrate to the native heart. - Biocompatible and functionalized gold nanowires (GNWs) are synthesized to decorate the intercellular microenvironment of the cardiac microtissues.
- In one or more embodiments, the GNWs were capped with polyethylene glycol bi-linker (COOH-PEG-SH) and functionalized using RGD cell adhesion motifs to 1) increase the biocompatibility of the conventional GNWs capped with toxic surfactant (Cetrimonium bromide, CTAB), 2) induce high negative surface charge to decrease the intracellular uptake, and 3) increase the GNW-cell interaction to enhance the spheroid structural integrity.
- Spheroid-based cardiac micro-tissues, with well defined geometrical features and mix functionalized GNWs with the micro-tissues are used to develop the final electrically conductive and injectable spheroid based cellular clusters.
- The procedure is minimally invasive, given that our injectable and electrically conductive micro-tissues can be directly delivered via catheter to the infarcted zone of myocardium (i.e., chest cavity not opened). In one or more embodiments, the micro-tissues can be delivered via a minimally invasive surgical catheter based delivery. The developed tissues could be used for in vivo testing in both small and large animal models.
- The preparation and functionalization of the GNWs can be conducted in a number of different manners. In one or more examples, preparation and functionalization of GNWs: and GNW-CTAB with average aspect ratio of 21 (˜1.1 μm length & ˜55 nm diameter) was synthesized. GNWs capped with SH-PEG-COOH (GNW-COOH) was synthesized via a customized 2-step exchange procedure including primary PEGylation in Tris buffer (
pH 3, 24 hr) followed by post-PEGylation in ethanol (20%, 24 hr). RGD functionalized GNWs (GNW-RGD) was synthesized using GNW-COOH and RGD peptide based on EDC/NHS protocol. Transmission electron microscopy, Raman spectroscopy and dynamic light scattering were used to characterize the produced GNWs (-CTAB, —COOH and -RGD). - Generation of cardiac micro-tissues: polydimethylsiloxane (PDMS) or Agarose concave microwells are used based on well-established protocols and our preliminary studies to engineer micro-tissues. Briefly, a micro-engineered master composed of an array of 20×20 concave microwells in the range of 100-300 μm diameter and 150 μm in spacing will be purchased or fabricated using SU-8 photoresist and soft lithography technique. Subsequently, a 10:1 mixture of elastomer and curing agent will be poured on PDMS master and racked out using a glass slide by applying light pressure. Surface tension will allow formation of meniscus on cylindrical microwells of PDMS master leading to formation of concave microwells. Cardiac cells mixed with functionalized GNWs having concentrations of 1, 10 and 50 micro-grams/ml will be seeded on microwells at a density of 10-15×10{circumflex over ( )}6 cells/ml of cardiac media. Passive cell seeding is expected to lead to formation of cellular clusters within each microwell. Upon cell seeding, microwells will be placed inside 37° C. for 7 days to form micro-tissues.
- In one or more embodiments, a unique and integrated strategy is proposed to develop next generation of MRT based on nanoengineering of scaffold-free cardiac microtissues using electrically conductive, cell adhesion-promoting and vasculogenic gold nanowires (GNWs) (
FIG. 1A ). Cardiomyocytes (CMs), functionalized on the cell surface, with GNWs that are conjugated with cell adhesion-promoting and vasculogenic or other peptides (FIG. 1B ), as the primary building blocks of the micro-tissues. Microscale technologies will be used to generate the scaffold-free tissues comprised of co-culture of functionalized CMs and cardiac fibroblast (CFs), with specific architectural arrangement (FIG. 1C ). In certain embodiments, the architectural arrangement may include a plurality of CFs arranged in at least one layer of fibroblasts that surrounds, encompasses, encircles, envelops, or encapsulates a cluster of CMs (e.g., functionalized CMs). In other embodiments, other architectural arrangements may be utilized. Since, gold nanomaterials-embedded scaffolds improve functional outcome in MRT, direct functionalization of CMs with multipurpose GNWs will allow scaffold-free MRT constructs that will lead to: a) establishment of mature cell-cell coupling due to GNWs that are conjugated with adhesion-promoting RGD peptide (FIG. 1D ); b) enhanced electromechanical integration (i.e. synchronous contraction) of the micro-tissues with the host myocardium due to high conductivity properties of GNWs (FIG. 1E ); and c) promotion of neovascular formation within and from the host towards the micro-tissues due to conjugation of VEGF-mimetic peptide to GNWs (FIG. 1F ). Addition of CFs, with specific architectural arrangement (i.e. within the outer layer of microtissues), will further enhance engraftment of the micro-tissues with the host while simultaneously promoting a native like endogenous niche through ECM production and paracrine CM-CF signaling. The proposed strategy produces injectable micro-tissues for intramyocardial delivery via minimally invasive catheter-based approach. - In some embodiments, scaffold-free, electrically conductive and vasculogenic cardiac micro-tissue include hiPSCs-CMs and CFs. In some embodiments, in vitro maturity and functionalities of micro-tissues including GNWs conjugated with RGD and VEGF-mimetic QK peptides is assessed. In some embodiments, scaffold-free cardiac micro-tissues including GNW-functionalized hiPSCs-CMs and CFs are generated for in vitro biological assessments.
- It is proposed that the functionalities of developed micro-tissues will work in an in vivo model, such as in preclinical rodent model and eventually in clinical applications. This will improve myocardial function, vascular formation and integration of micro-tissues within the host myocardium. The utility of the injected micro-tissues in improving myocardial function can be evaluated, and vascular formation and integration of the micro-tissues with the host myocardium are investigated.
- In one or more embodiments, functionalization of the surface of CMs with multipurpose GNWs are conjugated with cell adhesion promoting and vasculogenic peptides. Native ventricular myocardium consists of electrically conductive Purkinje fibers coupled with tightly packed cellular constructs consisting mainly of CMs and CFs. Previous work on the use of conductive nanomaterials (i.e. carbon nanotubes (CNTs), graphene oxide (GO), silicon (SO) nanowires, gold nanostructure) showed significant promise of these nanomaterials in enhanced functionalities of engineered cardiac tissues. However, most of these approaches relied on random dispersion of nanomaterials within scaffolding biomaterials (i.e. scaffold based approach) or cellular clusters without precise control over location or fate of the nanomaterials. Importantly, there are also potential cytotoxicity issues in the use of these nanomaterials (i.e. CNTs, GO, SO) for MRT. Nanoengineering is used to functionalize for the first time the surface of human induced pluripotent stem cell derived CMs (hiPSC-CMs), which will form the primary building blocks of the micro-tissues. For this, GNWs are used that are intrinsically electrically conductive, but in addition, the surface of GNWs are conjugated with cell adhesion-promoting RGD peptide (
FIG. 1A ). By providing nanoscale functionalization to promote cell-cell coupling (as a result of the presence of RGD peptide), as well as electromechanical integration with the surrounding host myocardium (as a result of the electrically conductive nature of GNWs) current limitations are addressed. - Neovascularization: Our approach is also designed to enhance neovascular formation by conjugation of vasculogenic VEGF-mimetic peptide (along with RGD), on the surface of GNWs. This strategy will address a significant limitation of current MRT approaches that lack vascular development to support and sustain implanted or injected engineered tissue constructs.
- Scaffold-free cardiac micro-tissues with specific architectural arrangement: In our unique strategy, microscale technology (i.e. use of thermo-responsive microwells) is used to develop scaffold-free cardiac micro-tissues comprised of co-culture of functionalized hiPSC-CMs and CFs with specific architectural arrangement. In particular, CFs are arranged within the outer layer of the micro-tissues with the primary purpose of promoting the engraftment of the micro-tissues with the host upon injection through cell-ECM interaction and production of native ECM proteins (i.e. collagen) by CFs. Our approach is the first strategy to generate cardiac micro-tissues with this unique architectural arrangement. Native myocardium is a multi-cellular and adaptive tissue consisting of myocytes and non-myocyte cells.
- The cellular mixture is significant as extensive studies show that the interaction between myocytes and non-myocytes, specifically CFs is an important component to maintain optimal functionalities of the myocardium through proper cellular signaling and gap junction proteins (i.e. Cx40, Cx43). Therefore, our unique strategy will lead to optimal functionalities of the microtissues through direct contact and paracrine CM-CF signaling. Although there may be resident CFs within the infarct zone thereby lessening the need for CFs in engineered micro-tissues, the majority of native CFs may be dysfunctional, are in activated pro-inflammatory state or already differentiated into myofibroblasts and may therefore not fully support the functionalities of injected CMs.
- It is important to note that our previous body of work and expertise in developing viable, functional, synchronously contracting engineered cardiac tissue using GNWs embedded gelatin-based scaffold (
FIG. 2A-2E ) and co-culture of CFs and CMs uniquely allow us to embark on this project and enhances its feasibility. - The embodiments herein develop this next generation of MRT, in totality, represents a conceptual and technological leap in innovation, rather than small incremental steps, from currently available technologies.
- The embodiments herein develop scaffold-free, electrically conductive and vasculogenic cardiac micro-tissues comprised of hiPSCs-CMs and CFs. Efficient MRT of infarcted myocardium relies on critical factors including proper cell source, enhanced retention and cell-cell coupling and engraftment, electromechanical integration of the engineered tissues with the surrounding host myocardium and neovascularization. We generate scaffold-free, and electrically conductive cardiac micro-tissues comprised of co-culture of hiPSCs-CMs and CFs with specific architectural arrangement for MRT. We will utilize nanoscale technology to synthesize GNWs and conjugate them with RGD and VEGF-mimetic peptides. We will then use a microengineering approach to establish micro-tissues with hiPSCs-CMs functionalized with GNWs comprising the core and CFs comprising the outer layer (
FIG. 1C ). We will perform extensive in vitro characterization to assess maturity, cellular connectivity, and electrophysiological functionalities of the micro-tissues. Electrically conductive scaffold-free micro-tissues will have enhanced cell-cell coupling as well as optimal physiological functionalities (synchronous contractility) in vitro. - The embodiments herein synthesize GNWs conjugated with RGD and VEGF-mimetic QK peptides GNWs, for example, with the average aspect ratio of 24 (˜5 μm in length and ˜50 nm in diameter) will be synthesized based on seed-mediated anisotropic growth method. We intend to start with 5 μm length for GNWs to reduce the likelihood of cellular uptake as previous studies have demonstrated the there is an inverse relation between the size and cellular uptake in gold nanomaterials. To conjugate RGD (cyclo(Arg-Gly-Asp-D-Phe-Cys) and VEGF-mimetic QK (Methacrylic acid-K(Ac)LTWQELYQLK(Ac)YK(Ac)GI-NH2 (SEQ ID NO: 1)) peptides on GNWs, first CTAB will be exchanged with COOH-PEGSH bi-linker; subsequently the peptides will be attached to the bi-linker via EDC/NHS procedure. The exchange of PEG bi-linker with CTAB will consist of two consecutive steps (
FIG. 3 ). First, CTAB-capped GNWs will be centrifuged (2000 rpm, 20 min) two times and re-dispersed in 1 mM CTAB solution to reduce the concentration of CTAB to the critical micelle formation concentration (CMC in water for CTAB is ˜1 mM). - After the second centrifugation/decantation, the supernatant will be discarded, and 400 μL of Tris buffer (50 mM, pH 3) will be added drop-wise to the GNWs pellet. To initiate the PEGylation process, 30 μL of COOH-PEG-SH (2 mM in DIW) will be added to the GNW-Tris mixture under vortexing and kept agitated for 1 min. The final mixture will be maintained undisturbed for 24 hr at room temperature to allow the completion of the PEGylation. The mixture will be then centrifuged (4000 rpm, 25 min) to remove the unreacted PEG bi-linkers, Tris buffer and free CTAB molecules. To further improve the CTAB-PEG bi-linker exchange, the freshly synthesized GNWPEG-COOH will be gently re-dispersed in 20% ethanol, followed by addition of 30 μL of COOH-PEG-SH (2 mM in 20% ethanol) to the mixture under gentle vortexing. The mixture will be kept undisturbed at room temperature for 24 hr. The two-step functionalized GNW-PEG-COOH will be harvested by centrifugation at 4000 rpm (25 min), and re-dispersed in 500 μL PBS.
- To conjugate RGD and QK peptides, the carboxylic acid groups of GNW-PEG-COOH will be activated by adding EDC and Sulfo-NHS forming the corresponding GNW-PEG-COO—NHS ester. Briefly, GNW-PEGCOOH, dispersed in 500 μL PBS (obtained from the previous step) will be added with 2 mg of EDC, vortex for 20 sec and then 5.5 mg of Sulfo-NHS will be added to this solution at room temperature for 10 min.
- Afterwards, 200 μL of 1:1 ratio of the peptides mixture (1 mg/mL in PBS each) will be added to the activated GNWs and the mixture will be kept in 4° C. for 5 hr. The GNW-PEG-RGD and GNW-PEG-QK mixture will be purified by centrifugation/decantation in PBS for 1 time. Upon preparation of GNWs and conjugation with RGD and VEGF-mimetic QK peptides, subsequent characterization will be performed using TEM, FTIR, Raman spectra and NMR. The presence and the size of GNWs will be obtained from TEM images. The conjugation of Au to the —SH group of HS-PEG-COOH will be characterized by the appearance of Au—S shift in Raman spectra. Furthermore, GNWs of the GNW-PEG-Peptides will be digested prior to taking NMR spectra. The appearance of amide I, amide II and amide A bands in the FTIR spectra and the appearance of the chemical shift corresponding to N—H bond in NMR spectra will confirm the presence and amount of peptides conjugated to GNWs.
- One or more embodiments herein will generate scaffold-free cardiac micro-tissues, using GNW-functionalized hiPSCs-CMs and CFs, and generate micro-tissues comprised of hiPSCs-CMs and CFs. CFs will be transfected to stably express yellow fluorescent proteins (CF-YFP) for tracking within the micro-tissues, while enabling 3-color imaging. An initial co-culture ratio of 3:1 (hiPSCs-CMs:CFs) will be selected based on our previous studies to enhance tissue-level function.
- To generate the micro-tissues, responsive PNIPAAam-based microwells (circular, starting with 250-300 μm diameter, 300 μm depth) along with twostep cell seeding process (hiPSCs-CMs followed by CFs, 3:1 ratio) (
FIG. 3 ). Briefly, hiPSCs-CMs (10×106 cells/ml in culture media) will be mixed with different concentrations of functionalized GNWs (0, 2.5 and 5 μg/ml) and will be seeded on the microwells at 25° C. The microwells will be rinsed in PBS to remove excess cells. Subsequently, microwells will be placed inside 37° C. for 2 h to increase surface area and volume, opening up room for seeding CFs (FIG. 4 ). Upon sequential seeding of both cell types, microwells will be placed inside 37° C. for 7 days for the formation of final micro-tissues. - One or more embodiments include visualization of the GNWs on cell surface. For example, upon 1, 2, and 3 weeks, tissue sectioning and TEM imaging are utilized to investigate the localization of GNWs on cell membrane within the micro-tissues. Briefly, microtissues are fixed in 2.5% glutaraldehyde and post-fixed in osmium tetroxide (1%) with 1.5% K+ ferricyanide. Fixed samples will then be dehydrated in ethanol/propylene oxide and embedded in epoxy resin for sectioning. Ultrathin slices (100 nm, Ultra microtome) are prepared and stained using uranyl acetate and lead citrate.
- In an example, assessment of cell-cell coupling within the micro-tissues is done as follows. On 1, 2, and 3 weeks of culture, the micro-tissues are fixed in 4% (v/v) paraformaldehyde (PF) in PBS and immunohistochemistry (IHC) consistent with our previous studies, will then be performed in each experimental condition (mono- and co-culture with defined concentrations of GNWs) to co-stain CX40, CX43 and CX45 gap junction proteins (Alexa Fluor 595, different samples) with sarcomeric proteins (MLC2a, MLC2v, α-actinin, α-MHC, Alexa Fluor 488). Within all the samples, the cells' nuclei will be stained with DAPI. The selection of secondary antibodies will enable 3-color imaging and identification of hiPSCs-CMs and YFP-CFs. We will quantify coverage area of the gap junction proteins within the selected regions of interest (ROIs, 300×300 μm2) as a function of GNWs concentrations, as well as the presence of CFs. Within different sets of samples we will use western blot to quantify the expression of gap junction proteins (CX40, CX43 and CX45). Briefly, micro-tissues in each experimental condition (mono- and co-culture with defined concentrations of GNWs) will be dissociated using AccuMax® solution (Innovative Cell Technologies). Cell suspension will be centrifuged at 1500 RPM for 5 min and supernatant removed. We will specifically look into the expression levels of CX40, CX43 and CX45. Extracted proteins will be ran through gel electrophoresis (SDS-PAGE). Proteins will be transferred to nitrocellulose membranes, and membranes treated with antibodies against corresponding cardiac markers and normalized against GAPDH. Densitometric analysis will be carried out with ImageJ software. In the co-culture condition, we will also assess the expression of secreted ECM proteins including laminin, fibronectin and collagen. Expected outcomes: Significantly higher area coverage and expression of gap junction proteins (Cx40/Cx43/Cx45) quantified by IHC and western blot in presence of GNWs and co-culture group with CFs.
- Contractility of the micro-tissues and sarcomere organization: The spontaneous contractility of the microtissues are assessed using real time video microscopy and a custom MATLAB code to quantify beating frequency (beats per min, BPM) and contractile signal patterns of the cells upon 1, 2, and 3 weeks consistent with our publications (n=3). Side-by-side of contractility analysis, we will utilize IHC images to analyze for sarcomere organization within the selected regions of interest (ROIs, 300×300 μm2) within each condition. Consistent to our previous work, this analysis will determine whether conjugated GNWs impart a significant enhancement on contractility of the micro-tissues correlated to sarcomere organization. Enhanced contractility of the micro-tissues (i.e. spontaneous beating synchrony) is expected within different ROIs, in groups functionalized with GNWs. Significantly increased alignment index of sarcomeres positively correlated with beating synchrony of the micro-tissues.
- Intracellular calcium (Ca2+) transients and electrophysiologic responsiveness of the micro-tissues: For Ca2+ transients measurements, samples will be exposed to 2.3 mM fluo-4 AM and 0.1% Pluronic F-127 for 15 min at 37° C. After 15 min, samples will be washed 3× in Tyrode's solution. Ca2+ transients will be imaged using confocal microscopy (63×) at 5-10 locations of each sample. Additionally, we will utilize qPCR analysis to assess upregulation of genes handling calcium proteins (i.e. SERCA2, RYR2, CACNA1C) in presence of GNWs. Within different samples, conduction velocity of cardiac micro-tissues will be analyzed using voltage sensitive dye RH-237. To determine contractile responsiveness to extraneous electrophysiologic signal (to mimic electrical signal from host myocardium), micro-tissues will be stimulated using platinum electrodes with 0.5-2 Hz, 5 V and 1-2 ms biphasic square pulses.
- Subsequently, action potential (AP) will be measured (5-10 locations) and conduction velocity quantified. Expected outcomes: Synchronized calcium (Ca2+) transients spikes across multiple ROIs of the microtissues as well as increased upregulation of genes handling calcium proteins as function in groups conjugated with GNWs. Increased level of action potential in co-culture groups with conjugated GNWs.
- Assessment of vascular formation: We utilized a microfluidic chip to assess utility of VEGF-mimetic QK peptide in promoting vascularization within 3D gelatin-based hydrogels. In brief, human umbilical vein endothelial cells (HUVECs, density: 15×106 cells/ml) were encapsulated within gelatin based hydrogel matrix (thiolated gelatin, Gel-S) conjugated with variable concentrations of QK mathacrylate peptide (sequence: KLTWQELYQLKYKGI-C (SEQ ID NO: 2), 0, 100, 150, 200 μg/mL). Our findings demonstrated that, in control condition (w/o QK), cells were randomly distributed throughout the matrix with disconnected and round morphologies. Alternatively, incorporation of QK peptide resulted in formation of robust, inter-connected vascular network with significantly increased average branch length, branch diameter and vascular network coverage. We will utilize a similar assay to encapsulate HUVECs along with micro-tissues (scaffold-free) within the microfluidic platform to assess the role of VEGF mimetic QK peptide, conjugated on GNWs, on vascular formation within the surrounding of the micro-tissues. Significant increase in ECs connectivity (network formation) and vascular area coverage toward the core of micro-tissues due to the presence of VEGF-mimetic QK peptide within the GNWs on the surface of hiPSCs-CMs. This will allow selection of optimal length and concentration of GNWs to proceed with in vivo studies.
- In one or more embodiments, if cellular uptake of GNWs occurs, we will adjust our synthesis process to increase the length to 7-10 μm. In case of cellular contraction and decrease in the size of micro-tissues to 150 μm, we will increase the diameter of the microwells (˜300-350 μm). In case, if we do not observe statistically significant difference in contractility or Ca2+ transients in presence of GNWs, we will optimize and increase the concentration in the range of 7.5 to 10 μg/ml.
- Embodiments further optionally include evaluating the functionalities of the developed cardiac micro-tissues in vivo in a rodent model (Months 16-24). Injectable cardiac micro-tissues, developed herein, are envisioned to significantly promote MRT through enhanced cell-cell coupling, cell-matrix interaction, electrical engraftment with the host myocardium and nonvascular formation. Therefore, it is important to evaluate biological and physiological performance of the injectable micro-tissue in vivo. In this, we will use small animal model (rodent) to study whether the developed cardiac micro-tissues will lead to such improved biological and physiological functionalities.
- The next generation of MRT is discussed herein, utilizing a multidisciplinary and state-of-the-art approach based on nanoengineered hiPSCs-CMs functionalized with multipurpose GNWs. The embodiments provide for the ultimate development of injectable micro-tissues with superior cell-cell coupling, engraftment, electromechanical integration and neovascularization leading to effective functional MTR.
- The gold nanostructures can be selected to optimize the utility of the resulting microtissue. Accordingly, the nature of the gold nanostructures is not critical, provided the resulting scaffold-free micro tissue possesses the desired physical properties and biological function. For example, the gold nanostructures can be 1D structures such as a wires, rods, or spheres. Typically the gold nanowires will have a width of about 20 nm to about 50 nm and a length of up to about 5 μm. Gold nanorods will typically have a width of about 50 nm and a length of up to about 100 nm, about 200 nm or about 300 nm. Gold nanospheres will typically have a diameter of up to about 50 nm or up to about 100 nm.
- The gold nanostructures can also include 2D structures such as nano-plates. Typically the gold nanoplates will be 2D sheets having dimensions up to about 250-500 nm×250-500 nm.
- The
gold nanostructures 100 can be linked to the cell-adhesion moiety or to the functional moiety through a direct chemical bond or through a linking group X, as shown inFIG. 6 . In one embodiment of the invention the gold nanostructure is linked to the cell-adhesion moiety or to the functional moiety through a direct chemical bond. In another embodiment of the invention the gold nanostructure is linked to the cell-adhesion moiety or to the functional moiety through a linking group X. Any linking group that provides a GNW with desired properties and function may be used. - In one embodiment of the invention the linker has a molecular weight of from about 20 daltons to about 20,000 daltons.
- In one embodiment of the invention the linker has a molecular weight of from about 20 daltons to about 5,000 daltons.
- In one embodiment of the invention the linker has a molecular weight of from about 20 daltons to about 1,000 daltons.
- In one embodiment of the invention the linker has a molecular weight of from about 20 daltons to about 200 daltons.
- In another embodiment of the invention the linker has a length of about 5 angstroms to about 60 angstroms.
- In another embodiment of the invention the linker separates the antigen from the remainder of the compound of formula I by about 5 angstroms to about 40 angstroms, inclusive, in length.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (—O—), and wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein one or more (e.g. 1, 2, 3, or 4) of the carbon atoms is optionally replaced by (—O—), and wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 25 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 2 to 10 carbon atoms.
- In another embodiment of the invention the linker is a divalent, branched or unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.
- In another embodiment of the invention the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 10 carbon atoms.
- In another embodiment of the invention the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 6 carbon atoms.
- In another embodiment of the invention the linker is a divalent, unbranched, saturated hydrocarbon chain, having from 2 to 4 carbon atoms.
- In another embodiment of the invention the linker comprises a polyethyleneoxy chain. In another embodiment of the invention the polyethyleneoxy chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating ethyleneoxy units.
- In another embodiment of the invention the linker is a divalent radical formed from a peptide.
- In another embodiment of the invention the linker is a divalent radical formed from an amino acid.
- In another embodiment of the invention the linker is a divalent radical of formula —S-(PEG)-C(═O)—, as illustrated in
FIG. 3 , wherein C(═O)— is bonded to the amino terminus of the RGD peptide. The molecular weight of PEG is 3500 Dalton g/mol. - The term “amino acid,” comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as unnatural amino acids (e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). The term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g. acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C1-C6)alkyl, phenyl or benzyl ester or amide; or as an α-methylbenzyl amide). Other suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T. W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981, and references cited therein). An amino acid can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine.
- The term “peptide” describes a sequence of 2 to 25 amino acids (e.g. as defined hereinabove) or peptidyl residues. The sequence may be linear or cyclic. For example, a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence. A peptide can be linked to the remainder of a compound of formula I through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine. Preferably a peptide comprises 3 to 25, or 5 to 21 amino acids. Peptide derivatives can be prepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; and 4,684,620, or as described in the Examples hereinbelow. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.
- The gold nanostructures can be linked to one or more cell adhesion moieties. The cell adhesion moiety provides affinity for targeting of the gold nanostructures toward the cell membrane. Additionally, these cell adhesion moieties are envisioned to enhanced cell-cell coupling among cardiomyocytes as well as cell cell-matrix interaction and overall cellular retention between the micro-tissues and the surrounding host matrix upon implantation. In one embodiment of the invention, since we are targeting the affinity of the gold nanostructures toward the cell membrane and aim to enhance the cell-cell coupling as well as cell cell-matrix interactions, the cell adhesion moiety would be a peptide, which is conjugated to the surface of the gold nanostructures. In another embodiment of the invention the cell adhesion moiety is an integrin binding peptides such as RGD or DGEA (SEQ ID NO: 3) or other peptides GRGDSP (SEQ ID NO: 4) or GRGDY (SEQ ID NO: 5) peptides. These peptides belong to classes of synthetic peptides that contain the amino acids: specifically for RGD: Arg-Gly-Asp, for DGEA (Asp-Gly-Glu-Ala) (SEQ ID NO: 3), for GRGDSP (H-Gly-Arg-Gly-Asp-Ser-Pro-OH) (SEQ ID NO: 4) and for GRGDY (Gly-Arg-Gly-Asp-Tyr) (SEQ ID NO: 5). (See Tissue Eng. 2000 April; 6(2):85-103, Extracellular Matrix Cell Adhesion Peptides: Functional Applications in Orthopedic Materials. LeBaron R G1, Athanasiou K A. See also Cook, A. D., Hrkach, J. S., Gao, N. N., Johnson, I. M., Pajvani, U. B., Cannizzaro, S. M., and Langer, R. Characterization and development of RGD-peptide-modified poly(lactic acid-co-lysine) as an interactive, restorable biomaterial; J. Biomed. Mater. Res. 35, 13, 1997.) These peptides will mimic cellular attachment activity. (See Pierschbacher, M. D., and Ruoslahti, E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309, 30, 1984. Pierschbacher, M. D., and Ruoslahti, E. Variants of the cell recognition site of fibronectin that retain attachment promoting activity. Proc. Natl. Acad. Sci. USA 81, 5985, 1984.) Other peptides can be used to promote cell adhesion through conjugation to the gold nanostructures.
- The gold nanostructures can be linked to one or more functional moieties. The functional moiety provides for specific target functionalities such as promoting vascular formation toward the injected tissues, reduce inflammatory response upon injection of the cells or reduce apoptosis. In one embodiment of the invention the functional moiety is a peptide. The function moiety could also be aptamers (oligonucleotides). In another embodiment of the invention the functional moiety is a vasculogenic peptide, an anti-inflammatory peptide, antiapoptotic/antinecrotic peptides. In one or more embodiments, the functional units and/or moieties includes one or more of vascularization peptides, anti-inflammatory peptides, antiapoptotic/antinecrotic peptides, or antioxidant peptides. For the vasculogenic peptides we can have: 1-QK (Methacrylic acid-K(Ac)LTWQELYQLK(Ac)YK(Ac)GI-NH2 (SEQ ID NO: 1)). (See Covalently immobilized VEGF-mimicking peptide with gelatin methacrylate enhances microvascularization of endothelial cells S P Parthiban, D Rana, E Jabbari, N Benkirane-Jessel, M Ramalingam Acta biomaterialia 51, 330-340). Or, VEGF memetic peptide [SLanc: K-(SL)3(RG)(SL)3-K-G-KLTWQE-LYQLKYKGI (SEQ ID NO: 6)]. (See Vivek A. Kumar, Nichole L. Taylor, Siyu Shi, Benjamin K. Wang, Abhishek A. Jalan, Marci K. Kang, Navindee C. Wickremasinghe, Jeffrey D. Hartgerink. Highly Angiogenic Peptide Nanofibers. ACS Nano, 2015, 9(1), 860-868.)
- The preparation of certain gold nanostructures that can be incorporated into the microtissues of the invention is illustrated in
FIG. 6 . For example, the gold nanostructure can be linked to a linking group (X), which can then be modified to incorporate an activating group (Y) that can be used to facilitate the attachment of the cell adhesion moiety or the functional moiety. Alternatively, the gold nanostructure can be linked directly to a group —X-Y in one step. Processes and reagents that can be used to modify a gold surface are known, for example, see Zhang, Z. & Lin, M. Fast loading of PEG-SH on CTAB-protected gold nanorods. RSC Adv. 4, 17760-17767 (2014), and/or Kinnear, C. et al. Gold Nanorods: Controlling Their Surface Chemistry and Complete Detoxification by a Two-Step Place Exchange. Angew. Chem. Int. Ed. 52, 1934-1938 (2013). - The linking group X can be attached to the gold nanostructure in any synthetically feasible linkage. In one embodiment, the gold surface can be attached to the linking group X through a covalent bond with a sulfur atom of X. The activating group Y can be a leaving group that can be displaced by an atom on the cell adhesion moiety or on the functional moiety—or the activating group Y can be a reactive group that can react with an atom on the cell adhesion moiety or on the functional moiety. In one embodiment, the activating group is sulfo N-hydroxysuccinamide (as illustrated in
FIG. 3 ) or another activated group that is capable of forming an amide with an amine group of the cell adhesion moiety or on the functional moiety. Such activated amide forming groups and reaction conditions are known. - The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
- Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Claims (19)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/740,071 US20220273845A1 (en) | 2017-10-11 | 2022-05-09 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762571056P | 2017-10-11 | 2017-10-11 | |
US16/157,956 US11364321B2 (en) | 2017-10-11 | 2018-10-11 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
US17/740,071 US20220273845A1 (en) | 2017-10-11 | 2022-05-09 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/157,956 Division US11364321B2 (en) | 2017-10-11 | 2018-10-11 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Publications (1)
Publication Number | Publication Date |
---|---|
US20220273845A1 true US20220273845A1 (en) | 2022-09-01 |
Family
ID=67844236
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/157,956 Active 2040-07-11 US11364321B2 (en) | 2017-10-11 | 2018-10-11 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
US17/740,071 Pending US20220273845A1 (en) | 2017-10-11 | 2022-05-09 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/157,956 Active 2040-07-11 US11364321B2 (en) | 2017-10-11 | 2018-10-11 | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Country Status (1)
Country | Link |
---|---|
US (2) | US11364321B2 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11364321B2 (en) * | 2017-10-11 | 2022-06-21 | Arizona Board Of Regents On Behalf Of Arizona State University | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11364321B2 (en) * | 2017-10-11 | 2022-06-21 | Arizona Board Of Regents On Behalf Of Arizona State University | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4612302A (en) | 1983-11-14 | 1986-09-16 | Brigham And Women's Hospital | Clinical use of somatostatin analogues |
US4684620A (en) | 1984-09-04 | 1987-08-04 | Gibson-Stephens Neuropharmaceuticals, Inc. | Cyclic polypeptides having mu-receptor specificity |
US4853371A (en) | 1986-06-17 | 1989-08-01 | The Administrators Of The Tulane Educational Fund | Therapeutic somatostatin analogs |
GB0524884D0 (en) | 2005-12-06 | 2006-01-11 | Syngenta Ltd | Improvements in or relating to organic compounds |
WO2013151755A1 (en) * | 2012-04-04 | 2013-10-10 | University Of Washington Through Its Center For Commercialization | Systems and method for engineering muscle tissue |
US20160106886A1 (en) * | 2013-05-20 | 2016-04-21 | Ramot At Tel-Aviv University Ltd | Metal-coated scaffolds for tissue engineering |
US10712339B2 (en) | 2014-10-01 | 2020-07-14 | Arizona Board Of Regents On Behalf Of Arizona State University | Engineering of a novel breast tumor microenvironment on a microfluidic chip |
US10017724B2 (en) | 2014-10-01 | 2018-07-10 | Arizona Board Of Regents On Behalf Of Arizona State University | Engineering of a novel breast tumor microenvironment on a microfluidic chip |
SG10201602911UA (en) * | 2015-04-14 | 2016-11-29 | Univ Singapore | Bioactive modification of poly(vinyl alcohol) with surface topography and biochemical cues for vascular graft |
US20170067025A1 (en) | 2015-09-02 | 2017-03-09 | Arizona Board Of Regents On Behalf Of Arizona State University | Tumor model for breast cancer cell migration studies and related methods |
US10265439B2 (en) | 2015-09-03 | 2019-04-23 | Arizona Board Of Regents On Behalf Of Arizona State University | Injectable cell-laden biohybrid hydrogels for cardiac regeneration and related applications |
US20170143871A1 (en) | 2015-10-29 | 2017-05-25 | Arizona Board Of Regents On Behalf Of Arizona State University | Gold Nanorod Incorporated Gelatin based Hybrid Hydrogels for Cardiac Tissue Engineering and Related Methods |
WO2018208782A1 (en) | 2017-05-09 | 2018-11-15 | Arizona Board Of Regents On Behalf Of Arizona State University | Non-fibrotic biocompatible electrode and related methods |
-
2018
- 2018-10-11 US US16/157,956 patent/US11364321B2/en active Active
-
2022
- 2022-05-09 US US17/740,071 patent/US20220273845A1/en active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11364321B2 (en) * | 2017-10-11 | 2022-06-21 | Arizona Board Of Regents On Behalf Of Arizona State University | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures |
Non-Patent Citations (2)
Title |
---|
Hussain et al. "Functional 3D Cardiac Co-Coculture Model Using Bioactive Chitosan Nanofiber Scaffolds" Biotechnology and Bioengineering, Vol 110, No. 2, February 2013 (Year: 2013) * |
Mei et al. "Recent Development in Therapeutic Cardiac Patches" Frontiers in cardiovascular Medicine 27 November 2020 (Year: 2020) * |
Also Published As
Publication number | Publication date |
---|---|
US20190275203A1 (en) | 2019-09-12 |
US11364321B2 (en) | 2022-06-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ju et al. | Extracellular vesicle-loaded hydrogels for tissue repair and regeneration | |
Ding et al. | Synthetic peptide hydrogels as 3D scaffolds for tissue engineering | |
US10245351B2 (en) | Malleable hydrogel hybrids made of self-assembled peptides and biocompatible polymers and uses thereof | |
Li et al. | Tissue engineering-based therapeutic strategies for vocal fold repair and regeneration | |
JP6224040B2 (en) | Modified self-assembling peptide | |
Hosseinkhani et al. | Self-assembled proteins and peptides for regenerative medicine | |
Reis et al. | A peptide-modified chitosan–collagen hydrogel for cardiac cell culture and delivery | |
Jain et al. | Controlling neuronal cell growth through composite laminin supramolecular hydrogels | |
US20110280914A1 (en) | Hydrogels crosslinked with gold nanoparticles and methods of making and using thereof | |
Kinikoglu et al. | The influence of elastin-like recombinant polymer on the self-renewing potential of a 3D tissue equivalent derived from human lamina propria fibroblasts and oral epithelial cells | |
KR20160091993A (en) | Self-assembling peptides, peptidomimetics and peptidic conjugates as building blocks for biofabrication and printing | |
KR20160088431A (en) | Novel ultrashort hydrophobic peptides that self-assemble into nanofibrous hydrogels and their uses | |
Tsao et al. | Electrospun patch functionalized with nanoparticles allows for spatiotemporal release of VEGF and PDGF-BB promoting in vivo neovascularization | |
Vandghanooni et al. | Natural polypeptides-based electrically conductive biomaterials for tissue engineering | |
Wang et al. | The study of angiogenesis stimulated by multivalent peptide ligand-modified alginate | |
CN110753537A (en) | Non-covalently assembled conductive hydrogels | |
US20220273845A1 (en) | Nano scale decoration of scaffold-free microtissue using functionalised gold nanostructures | |
Li et al. | Enzymatically functionalized RGD-gelatin scaffolds that recruit host mesenchymal stem cells in vivo and promote bone regeneration | |
Gao et al. | Advances in cell membrane-encapsulated biomaterials for tissue repair and regeneration | |
Guan et al. | Dual-bionic regenerative microenvironment for peripheral nerve repair | |
Koutsopoulos | Self-assembling peptides in biomedicine and bioengineering: tissue engineering, regenerative medicine, drug delivery, and biotechnology | |
US20210322628A1 (en) | Functional amyloid hydrogels and applications thereof | |
Liu et al. | Presentation of bioactive epitopes with free N-termini on self-assembling peptide nanofibers | |
Sezer et al. | Peptide-Based Bioink Development for Custom-made Bioprinter with Specialized Nozzle Design | |
Broguiere | Engineering of synthetic extracellular matrix analogues supporting and guiding neurons |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY, ARIZONA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NIKKHAH, MEHDI;NAVAEI, ALI;SIGNING DATES FROM 20181220 TO 20181221;REEL/FRAME:059984/0918 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
AS | Assignment |
Owner name: THE UNITED STATES OF AMERICA AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS, DISTRICT OF COLUMBIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:MIGRINO, RAYMOND;REEL/FRAME:060126/0048 Effective date: 20210101 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |