AU2016262958A1 - Surface treatments - Google Patents
Surface treatments Download PDFInfo
- Publication number
- AU2016262958A1 AU2016262958A1 AU2016262958A AU2016262958A AU2016262958A1 AU 2016262958 A1 AU2016262958 A1 AU 2016262958A1 AU 2016262958 A AU2016262958 A AU 2016262958A AU 2016262958 A AU2016262958 A AU 2016262958A AU 2016262958 A1 AU2016262958 A1 AU 2016262958A1
- Authority
- AU
- Australia
- Prior art keywords
- dope
- integer
- construct
- lipid
- substituent
- 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.)
- Abandoned
Links
- 238000004381 surface treatment Methods 0.000 title claims description 17
- 238000000034 method Methods 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 229910001868 water Inorganic materials 0.000 claims abstract description 30
- 238000002360 preparation method Methods 0.000 claims description 18
- 125000001931 aliphatic group Chemical group 0.000 claims description 14
- 239000006185 dispersion Substances 0.000 claims description 14
- 229920000768 polyamine Polymers 0.000 claims description 14
- 150000001768 cations Chemical class 0.000 claims description 12
- -1 petroselenyl Chemical group 0.000 claims description 12
- 125000001424 substituent group Chemical group 0.000 claims description 10
- 125000002252 acyl group Chemical group 0.000 claims description 7
- 229920006395 saturated elastomer Polymers 0.000 claims description 5
- 125000003342 alkenyl group Chemical group 0.000 claims description 3
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000003475 gondoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000002811 oleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000001236 palmitoleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 12
- 239000002245 particle Substances 0.000 abstract description 4
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- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 45
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 27
- 239000000203 mixture Substances 0.000 description 27
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 24
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- 230000000845 anti-microbial effect Effects 0.000 description 17
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- 229910001220 stainless steel Inorganic materials 0.000 description 15
- 239000010935 stainless steel Substances 0.000 description 15
- 150000001875 compounds Chemical class 0.000 description 14
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 13
- 229960001701 chloroform Drugs 0.000 description 13
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 125000006367 bivalent amino carbonyl group Chemical group [H]N([*:1])C([*:2])=O 0.000 description 10
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 10
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- 239000002953 phosphate buffered saline Substances 0.000 description 10
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- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 238000005481 NMR spectroscopy Methods 0.000 description 9
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- 229910002027 silica gel Inorganic materials 0.000 description 9
- 238000009472 formulation Methods 0.000 description 8
- 241000191967 Staphylococcus aureus Species 0.000 description 7
- 230000003115 biocidal effect Effects 0.000 description 7
- 150000002148 esters Chemical class 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 7
- RFCIORHQRSVYDS-UHFFFAOYSA-N 2-[(2-methoxy-2-oxoethyl)-[2-[[2-[(2-methylpropan-2-yl)oxycarbonylamino]acetyl]amino]acetyl]amino]acetic acid Chemical compound COC(=O)CN(CC(O)=O)C(=O)CNC(=O)CNC(=O)OC(C)(C)C RFCIORHQRSVYDS-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical group CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 6
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000004599 antimicrobial Substances 0.000 description 6
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- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000007943 implant Substances 0.000 description 6
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- 238000012546 transfer Methods 0.000 description 6
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- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 5
- 239000012043 crude product Substances 0.000 description 5
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 5
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 5
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
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- 125000002091 cationic group Chemical group 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
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- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical group OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 4
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- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 description 3
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- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical group [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 206010065042 Immune reconstitution inflammatory syndrome Diseases 0.000 description 1
- 238000006845 Michael addition reaction Methods 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- 206010067268 Post procedural infection Diseases 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- HIHOWBSBBDRPDW-PTHRTHQKSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 description 1
- 229940022682 acetone Drugs 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
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- 238000004220 aggregation Methods 0.000 description 1
- 125000002723 alicyclic group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000010065 bacterial adhesion Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 229940076134 benzene Drugs 0.000 description 1
- WDODWFPDZYSKIA-UHFFFAOYSA-N benzeneselenol Chemical compound [SeH]C1=CC=CC=C1 WDODWFPDZYSKIA-UHFFFAOYSA-N 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 230000005587 bubbling Effects 0.000 description 1
- 125000006355 carbonyl methylene group Chemical group [H]C([H])([*:2])C([*:1])=O 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- WGXZDYPGLJYBJW-UHFFFAOYSA-N chloroform;propan-2-ol Chemical compound CC(C)O.ClC(Cl)Cl WGXZDYPGLJYBJW-UHFFFAOYSA-N 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- FMJJKGCANRBYNT-UHFFFAOYSA-L dipotassium dioxido(selanylidene)-lambda4-sulfane Chemical compound S(=[Se])([O-])[O-].[K+].[K+] FMJJKGCANRBYNT-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000000635 electron micrograph Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
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- 210000003890 endocrine cell Anatomy 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000011905 homologation Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000000854 inhibitional effect Effects 0.000 description 1
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- 239000013067 intermediate product Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
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- 150000002611 lead compounds Chemical class 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- VMVNZNXAVJHNDJ-UHFFFAOYSA-N methyl 2,2,2-trifluoroacetate Chemical compound COC(=O)C(F)(F)F VMVNZNXAVJHNDJ-UHFFFAOYSA-N 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- LSHROXHEILXKHM-UHFFFAOYSA-N n'-[2-[2-[2-(2-aminoethylamino)ethylamino]ethylamino]ethyl]ethane-1,2-diamine Chemical compound NCCNCCNCCNCCNCCN LSHROXHEILXKHM-UHFFFAOYSA-N 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 125000001117 oleyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 150000002898 organic sulfur compounds Chemical class 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 230000002138 osteoinductive effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- ORQWTLCYLDRDHK-UHFFFAOYSA-N phenylselanylbenzene Chemical compound C=1C=CC=CC=1[Se]C1=CC=CC=C1 ORQWTLCYLDRDHK-UHFFFAOYSA-N 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- IZLFUDHPNDYYHS-UHFFFAOYSA-M potassium;2-bromoacetate Chemical compound [K+].[O-]C(=O)CBr IZLFUDHPNDYYHS-UHFFFAOYSA-M 0.000 description 1
- KYEKHFSRAXRJBR-UHFFFAOYSA-M potassium;selenocyanate Chemical compound [K+].[Se-]C#N KYEKHFSRAXRJBR-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 229930195734 saturated hydrocarbon Natural products 0.000 description 1
- SPVXKVOXSXTJOY-UHFFFAOYSA-N selane Chemical compound [SeH2] SPVXKVOXSXTJOY-UHFFFAOYSA-N 0.000 description 1
- 229910000058 selane Inorganic materials 0.000 description 1
- CRDYSYOERSZTHZ-UHFFFAOYSA-N selenocyanic acid Chemical class [SeH]C#N CRDYSYOERSZTHZ-UHFFFAOYSA-N 0.000 description 1
- 150000003346 selenoethers Chemical class 0.000 description 1
- 239000013545 self-assembled monolayer Substances 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 125000002328 sterol group Chemical group 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003463 sulfur Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- OJNHYHSAGMVROG-UHFFFAOYSA-N tert-butyl (2-methylpropan-2-yl)oxycarbonyl carbonate methyl 2,2,2-trifluoroacetate Chemical compound COC(=O)C(F)(F)F.CC(C)(C)OC(=O)OC(=O)OC(C)(C)C OJNHYHSAGMVROG-UHFFFAOYSA-N 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
- A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pest Control & Pesticides (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Agronomy & Crop Science (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Methods of treating surfaces to provide an antibacterial surface or a surface to which micro-dimensioned particles will adhere are disclosed. Water dispersible selenide-lipid and polycation-lipid constructs for use in the methods are also disclosed.
Description
SURFACE TREATMENTS
TECHNICAL FIELD
The invention relates to methods of treating surfaces to provide advantageous properties, e.g. antimicrobial properties, and lipid constructs for use in such methods. In particular, the invention relates to methods of treating surfaces using selenide-lipid and polyamine-lipid constructs.
BACKGROUND ART
As stated in the publication of Gallo et al (2014) it is expected that the projected increased usage of implantable devices in medicine will result in a natural rise in the number of infections related to these cases. The current knowledge of antimicrobial surface treatments suitable for prevention of infection is reviewed. Surface treatment modalities include minimizing bacterial adhesion, biofilm formation inhibition and bactericidal action.
The publications of Reid and others disclose biocidal formulations including a selenium (Se) compound. The selenium compounds may be deposited on a surface and covalently or non-covalently associated with it. A broad range of selenium compounds are proposed, including compounds of the formula RSeX where R is an aliphatic or phenolic group and X is a protecting group.
Cationic lipids have primarily been developed for use in liposomal gene delivery as an alternative to viral-based gene delivery, but have also been identified as having bactericidal activity. Common cationic lipid classes include N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 3β [N-(Ν', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol). At least partly because of the low efficiency of lipofection the vast majority of clinical trials of gene therapy have used alternative means of gene delivery. The further development of cationic lipids has sought to improve the efficiency of lipofection.
The publications of Behr et al (1989) and Remy et al (1994) disclose spermine-lipid conjugates where the lipid is a phosphatidylethanolamine (DOPES and DPPES). Conjugation is via the carboxyl function of a functionalised L-5-carboxyspermine derivative. The conjugates are used in the preparation of compacted lipopolyamine-coated plasmids . The coated plasmids are used in a transfection procedure.
The publication of Byk et al (1989) discloses structure-activity relationships amongst a series of cationic-lipids developed for use in DNA transfer. Amongst the lipoamines evaluated in these studies, the polyamine geometry was shown to have an influence on transfection efficiency.
The publication of Randazzo et al (2009) discloses an exploration of the dual functionality of cationic lipids in the context of gene transfer and bactericidal activity. The cationic lipids demonstrated to possess these activities comprised a sterol moiety as the lipid component.
The publication of Kato et al (2003) discloses a method of adhering otherwise non-adherent cells to surfaces using a biocompatible anchor.
It is an object of the present invention to provide a method of treating the surface of surgical dressings and implants using lipid constructs that is effective to reduce the incidence of postoperative infections. It is an object of the present invention to provide a method of treating a surface that is effective to promote the adherence of micro-dimensioned particles to the surface. It is an object of the present invention to provide selenium and polycation-lipid constructs for use in these methods. These objects are to be read in the alternative with the object at least to provide a useful choice in the selection of such methods or constructs .
DISCLOSURE OF INVENTION
In a first aspect the invention provides an antimicrobial surface treatment method comprising the step of contacting the surface of an object with an aqueous dispersion of at least one functional-lipid construct where the lipid is a di-acyl, di-alkenyl or di-alkyl glycerophospholipid and the functional moiety of the construct confers the antimicrobial activity.
Preferably, the object is a surgical dressing or implant. More preferably, the object is a surgical implant. Most preferably, the surface is stainless steel.
Preferably, the aqueous dispersion is devoid of detergents and organic solvents. More preferably, the aqueous dispersion consists of water and the at least one functional-lipid construct.
Preferably, the lipid is a di-acyl glycerophospholipid. More preferably, the lipid is a phosphatidylethanolamine. Most preferably, the lipid is a di-oleoyl phosphatidylethanolamine.
Preferably, the functional moiety is selected from the group consisting of: selenide and polycation. More preferably, the functional moiety is selected from the group consisting of: cyanoselenide and polyamine. Most preferably, the functional moiety is cyanoselenide or spermine.
Preferably, the antimicrobial surface treatment is an antibacterial surface treatment. More preferably, the antimicrobial surface treatment is a bactericidal surface treatment.
Preferably, the contacting the surface is by immersing the object in the dispersion for a time sufficient to provide the antimicrobial surface treatment. More preferably, the time is less than 60 seconds. Yet more preferably, the time is less than 30 seconds. Most preferably, the time is less than 10 seconds.
Preferably, the dispersion is sonicated whilst the object is immersed.
Preferably, the concentration of the construct in the dispersion is sufficient to provide the antimicrobial surface treatment. More preferably, the concentration is less than 1 mg/mL of construct.
In a first embodiment of the first aspect the invention provides an antimicrobial surface treatment method comprising the step of contacting the surface with an aqueous dispersion of a selenide-lipid construct where the lipid is a di-acyl, di-alkenyl or di-alkyl glycerophospholipid. Preferably, the lipid is a diacylglycerolipid. More preferably, the lipid is a diacyl-glycerophospholipid. Most preferably, the lipid is phosphatidylethanolamine.
In a second embodiment of the first aspect the invention provides a method of treating the surface of a surgical implant comprising the step of contacting the surface with an aqueous dispersion of a polycation-lipid construct where the polycation is an N1-acylated polyamine and the lipid is a di-acyl, dialkenyl or di-alkyl glycerophospholipid. Preferably, the lipid is a diacylglycerolipid. More preferably, the lipid is a diacylglycerophospholipid. Most preferably, the lipid is phosphatidylethanolamine.
In the second embodiment the polycation-lipid construct is preferably of the structure :
where M is a monovalent cation, n is the integer 3, 4 or 5 and X is the divalent radical methylene (-CH2-), Ri and R2 are independently selected from the group consisting of C14-20 saturated, mono- or di- unsaturated, unbranched acyl groups, and R3 is the N1-acylated polyamine.
In a second aspect the invention provides a selenide-lipid construct of the structure :
where : m is the integer 1,2,3 or 4; preferably the integer 1, 2 or 4; most preferably the integer 2; n is the integer 3, 4 or 5; most preferably the integer 4; p is the integer 1, 2 or 3; most preferably the integer 2; q is the integer 1, 2 or 3; most preferably the integer 1; M is a monovalent substituent; preferably the monovalent substituent CH3 or H; most preferably the monovalent substituent H; M' is a monovalent cation or substituent; preferably the monovalent cation H+, K+ or Na+; most preferably the monovalent cation H+; and
Ri and R2 are independently an aliphatic C14-20 acyl, aliphatic C14-20 alkenyl or aliphatic C14-20 alkyl substituent; preferably a substituent selected from the group consisting of myristyl, palmityl, stearyl, arachidyl, palmitoleoyl, petroselenyl, oleoyl, elaidyl, vaccenyl and gondoyl; most preferably the aliphatic Cis alkenyl substituent oleoyl.
In a third aspect the invention provides a polycation-lipid construct of the structure :
where X is -CH2- and n is the integer 3, 4 or 5; Ri and R2 are independently selected from the group consisting of aliphatic C14-20 acyl, aliphatic C14-20 alkenyl or aliphatic C14-20 alkyl; and R3 is an N1-acylated polyamine.
Preferably, Ri and R2 are independently selected from the group consisting of: C14-20 acyl groups that are unbranched and saturated or mono-unsaturated. More preferably, Ri and R2 are independently selected from the group consisting of: myristyl, palmityl, stearyl, arachidyl, palmitoleoyl, petroselenyl, oleoyl, elaidyl, vaccenyl and gondoyl. Most preferably, Ri and R2 are the aliphatic Cis alkenyl substituent oleoyl.
Preferably, R3 is of the structure:
or
In a fourth aspect the invention provides a surface treatment preparation consisting essentially of a dispersion in water of at least one construct of the second or third aspects of the invention.
In the description and claims of this specification the following acronyms, terms and phrases have the meaning provided: "alicyclic" means cyclic aliphatic; "aliphatic" means alkanes, alkenes or alkynes or their derivatives and is used as a descriptor for compounds that do not have the special stability of aromatics; "alkanes" means a saturated hydrocarbon of the general formula CnH2n+2; "alkenes" means unsaturated hydrocarbons that contain one or more double carbon-carbon bonds; "alkynes" means unsaturated hydrocarbons that contain one or more triple carbon-carbon bonds; "aromatic" means containing a benzene ring or having similar chemical properties; "Boc" means tert-butoxycarbonyl; "BocsSpm" means (N1, N4, N9-tri-tert-butoxycarbonyl) -1,12-diamino-4,9-diazadodecane; "comprising" means "including", "containing" or "characterized by" and does not exclude any additional element, ingredient or step; "consisting essentially of" means excluding any element, ingredient or step that is a material limitation; "consisting of" means excluding any element, ingredient or step not specified except for impurities and other incidentals; "dispersible in water" means dispersible in pure, deionised water at 25 °C in the absence of organic solvents or surfactants to provide a dispersion at a concentration of at least 1 μπιοΙ/πΛ and "water dispersible" has a corresponding meaning; "DOPE" means 1,2-0-dioleoyl-sn-glycero-3-phosphatidylethanolamine; "DSPE" means 1,2-0-distereoyl-sn-glycero-3-phosphatidylethanolamine; "hydrophilic" means having a tendency to mix with, dissolve in, or be wetted by water and "hydrophilicity" has a corresponding meaning; "hydrophobic" means having a tendency to repel or fail to mix with water and "hydrophobicity" has a corresponding meaning; "monovalent cation" means an ion having a single positive charge and includes the monovalent cations H+, Na+, K+ or (CH3CH2)3N+; "N1-acylation" means the attachment of an acyl group (RCO-) at a terminal, primary amine of the longest chain of the molecule and "N1-acylated" has a corresponding meaning; "polyamine" means an unbranched organic compound comprising three or more amine functions including at least two primary amino (-11¾) functions; and "Spm" (or "spm") means spermine .
The terms "first", "second", "third", etc. used with reference to elements, features or integers of the subject matter defined in the Statement of Invention and Claims, or when used with reference to alternative embodiments of the invention are not intended to imply an order of preference.
Where concentrations or ratios of reagents are specified the concentration or ratio specified is the initial concentration or ratio of the reagents. Where values are expressed to one or more decimal places standard rounding applies. For example, 1.7 encompasses the range 1.650 recurring to 1.749 recurring.
In the absence of further limitation, the use of plain bonds in the representations of the structures of compounds encompasses the diastereomers, enantiomers and mixtures thereof of the compounds. In the representations of the structures or substructures of compounds the repeat of a divalent radical is represented by:
where -X- is the divalent radical repeated n times . Where the divalent radical is methylene (-CH2-) the repeat of this divalent radical is represented by:
To facilitate the description of the preparation and use of the constructs the following designations are used: "-Ad-" designates the substructure:
or
where n is the integer 4; "-CMG(m)-" designates the substructure:
where m is the integer 1, 2, 3 or 4 and M is a monovalent substituent; and "-DOPE" designates the substituent of the structure:
or
where M' is a monovalent cation (typically H+) .
The invention will now be described with reference to embodiments or examples and the figures of the accompanying drawings pages .
BRIEF DESCRIPTION OF DRAWINGS
Figure 1. ΧΗ NMR spectrum of the cyanoselenide-lipid construct designated NCSeCH2CO-CMG(2)-Ad-DOPE
Figure 2. Fluorescence microscopy of the surface of untreated (A) and treated (B) coupons following incubation in the presence of viable cultures of Staphylococcus aureus.
Figure 3. Fluorescence microscopy of the surface of untreated (A) and treated (B) coupons following incubation in the presence of viable cultures of Staphylococcus epidermis.
Figure 4. Photographs of incubated blood agar plates following inoculation with cultures of Staphylococcus aureus exposed to untreated (A) and treated (B) coupons .
Figure 5. Photographs of incubated blood agar plates following inoculation with cultures of Staphylococcus epidermis exposed to untreated (A) and treated (B) coupons.
Figure 6. Scanning electron micrographs (350x) of samples of untreated (A) and treated (B) surgical dressing using the construct designated NCSeCEhCO-CMG(2)-Ad-DOPE in the treatment.
Figure 7. Scanning electron micrographs (3,500x) of samples of untreated (A) and treated (B) surgical dressing using the construct designated NCSeCEhCO-CMG(2)-Ad-DOPE in the treatment.
Figure 8. Scanning electron micrographs (4,500x) of surfaces of stainless steel squares treated with the construct designated NCSeCH2C0-CMG(2)-Ad-DOPE (A) , the construct designated HA-gar-Ad-DOPE (B), the construct designated Spm-Ad-DOPE (C) or untreated (D) and incubated in the presence of Staphylococcus aureus.
Figure 9. Scanning electron micrographs (4,500x) of surfaces of stainless steel squares treated with the construct designated NCSeCH2CO-CMG(2)-Ad-DOPE (A) , the construct designated HA-gar-Ad-DOPE (B), the construct designated Spm-Ad-DOPE (C) or untreated (D) and incubated in the presence of Staphylococcus epidermis.
Figure 10. Scanning electron micrographs at the magnifications indicated in each micrograph of the surface of Capture-R™ Ready-Id (A), Capture-R™ Ready-
Screen (B), CT-6 (C) , Spm-Ad-DOPE (9a) treated and fixed (D), Spm-Ad-DOPE (9a) treated and lysed (E) and untreated (F) plates.
Figure 11. Photomicrographs (lOOx) of surfaces treated with Spm-Ad-DOPE (9a) in either PBS (A) or SucT (B) or spermine (C) following contact with a suspension of RBCs.
Figure 12. Scanning electron micrographs of the surface of untreated (A) and treated (B) laminated nylon mesh following exposure to particulates generated by a wood burner.
DESCRIPTION OF EMBODIMENTS
The method of the invention provides a convenient biocompatible means of treating surgical dressings and implants at the location and time of use by clinicians and surgeons .
Cyanoselenide as the functional moiety
The preparation of the constructs designated Mai-(CH2) 2CO-CMG(2)-Ad-DOPE and H-CMG(2)-Ad-DOPE is disclosed in the publication of Bovin et al (2008) and restated here for the sake of completeness. Acetone, benzene, chloroform, ethylacetate, methanol, toluene and o-xylene were from Chimmed (Russian Federation). Acetonitrile was from Cryochrom (Russian Federation). DMSO, DMF, CF3COOH, Et3N, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide were from Merck (Germany). Iminodiacetic acid dimethyl ester hydrochloride was from Reakhim (Russian Federation). Dowex 50X4-400 and Sephadex LH-20 were from Amersham Biosciences AB (Sweden). Silica gel 60 was from Merck (Germany) . Tetraamine (PRN-CEh^C x 2H2SO4 was synthesized as described by Litherland et al. (1938). Thin-layer chromatography was performed using silica gel 60 F254 aluminium sheets (Merck, 1.05554) with detection by charring after 7% H3PO4 soaking.
Preparation of {[2- (2-tert-hutoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester
To a stirred solution of (methoxycarbonylmethyl-amino)-acetic acid methyl ester hydrochloride (988 mg, 5 mmol) in DMF (15 ml) were added Boc-GlyGlyNos (3293 mg, 10 mmol) and (CHsCERJsN (3475 μ]3, 25 mmol) were added. The mixture was stirred overnight at room temperature and then diluted with o-xylene (70 ml) and evaporated. Flash column chromatography on silica gel (packed in toluene, and eluted with ethyl acetate) resulted in a crude product. The crude product was dissolved in chloroform and washed sequentially with water, 0.5 M NaHC03 and saturated KC1. The chloroform extract was evaporated and the product purified on a silica gel column (packed in chloroform and eluted with 15:1 (v/v) chloroform/methanol). Evaporation of the fractions and drying under vacuum of the residue provided a colourless thick syrup. Yield 1785 mg, (95%) . TLC: R.f=0.49 (7:1 (v/v) chlorof orm/methanol) . ΧΗ NMR (500 MHz, [D6]DMSO, 30 °C) δ, ppm: 7.826 (t, J=5.1 Hz, 1H; NHCO), 6.979 (t, J=5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH2COO), 3.969 (d, J=5.1 Hz, 2H; COCH2NH) , 3.689 and 3.621 (s, 3H; OCH3) , 3.559 (d, J=5.9 Hz, 2H; COCH2NHCOO) , 1.380 (s, 9H; C(CH3) 3) .
Preparation of {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid
To a stirred solution of { [2-(2-tert-butoxycarbonylamino-acetylamino) -acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2 M aqueous NaOH (23.5 ml) was added and the solution kept for 5 min at room temperature. The solution was then acidified with acetic acid (0.6 ml) and evaporated to dryness. Column chromatography of the residue on silica gel (packed in ethyl acetate and eluted with 2:3:1 (v/v/v) i-PrOH/ethyl acetate/water) resulted in a recovered { [2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester (63 mg, 3.4%) and target compound (1320 mg). The intermediate product was then dissolved in methanol/water/pyridine mixture (20:10:1, 30 ml) and passed through an ion exchange column (Dowex 50X4-400, pyridine form, 5 ml) to remove residual sodium cations. The column was then washed with the same solvent mixture, the eluant evaporated, the residue dissolved in chloroform/benzene mixture (1:1, 50 ml) and then evaporated and dried under vacuum. Yield of 10 was 1250 mg (74%), white solid. TLC: Rf=0.47 (4:3:1 (v/v/v) i-PrOH/ethyl acetate/water). XH NMR (500 MHz, [De]DMSO, 30 °C), mixture of cis- and trans- conformers of N-carboxymethylglycine unit c.3:l. Major conformer; δ, ppm: 7.717 (t, J=5 Hz, 1H; NHCO), 7.024 (t, J=5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH2COOCH3) , 3.928 (d, J=5 Hz, 2H; COCH2NH) , 3.786 (s, 2H; NCH2COOH), 3.616 (s, 3H; OCH3) , 3.563 (d, J=5.9 Hz, 2H; COCH2NHCOO), 1.381 (s, 9H; 0(0¾) 3) ppm; minor conformer, δ = 7.766 (t, J= 5 Hz, 1H; NHCO), 7.015 (t, J=5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH2COOCH3) , 3.928 (d, J=5 Hz, 2H; COCH2NH) , 3.858 (s, 2H; NCH2COOH), 3.676 (s, 3H; OCH3) , 3.563 (d, J=5.9 Hz, 2H; COCH2NHCOO), 1.381 (s, 9H; 0(0Η3)3).
Preparation of {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid N-oxysuccinimide ester (Boc-Gly2 (MCMGly) Nos)
To an ice-cooled stirred solution of {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid (1200 mg, 3.32 mmol) and iV-hydroxysuccinimide (420 mg, 3.65 mmol) in DMF (10 ml) was added N, Ν'-dicyclohexylcarbodiimide (754 mg, 3.65 mmol). The mixture was stirred at 0°C for 30 min, then for 2 hours at room temperature. The precipitate of N,N'~ dicyclohexylurea was filtered off, washed with DMF (5 ml), and filtrates evaporated to a minimal volume. The residue was then agitated with (CH3CH2)20 (50 ml) for 1 hour and an ether extract removed by decantation. The residue was dried under vacuum providing the active ester (1400 mg, 92%) as a white foam. TLC: 13^=0.71 (40:1 (v/v) acetone/acetic acid) . ΧΗ NMR (500 MHz, [De]DMSO, 30 °C), mixture of cis- and trans- conformers of N-carboxymethylglycine unit c. 3:2.
Major conformer; δ, ppm: 7.896 (t, J=5.1 Hz, 1H; NHCO), 6.972 (t, J=5.9 Hz, 1H; NHCOO), 4.533 (s, 2H; NCH2COON), 4.399 (s, 2H; NCH2COOCH3) , 3.997 (d, J=5.1 Hz, 2H; COCH2NH) , 3.695 (s, 3H; OCH3), 3.566 (d, J=5.9 Hz, 2H; COCH2NHCOO), 1.380 (s, 9H; C(CH3)3).
Minor conformer; δ, ppm: 7.882 (t, J=5.1 Hz, 1H; NHCO), 6.963 (t, J=5.9 Hz, 1H; NHCOO), 4.924 (s, 2H; NCH2COON), 4.133 (s, 2H; NCH2COOCH3) , 4.034 (d, J=5.1 Hz, 2H; COCH2NH) , 3.632 (s, 3H; OCH3), 3.572 (d, J=5.9 Hz, 2H; COCH2NHCOO) , 1.380 (s, 9H; C(CH3) 3) .
The active ester (1380 mg) was dissolved in DMSO to provide a volume of 6 ml and used as a 0.5 M solution (stored at -18 °C).
Preparation of {[2- (2-tert-hutoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester
To the stirred solution of (methoxycarbonylmethyl-amino)-acetic acid methyl ester hydrochloride (988 mg, 5 mmol) in DMF (15 ml) Boc-GlyGlyNos (3293 mg, 10 mmol) and Et3N (3475 μΐ, 25 mmol) were added. The mixture was stirred overnight at room temperature (r.t.), then diluted with o-xylene (70 ml) and evaporated. Flash column chromatography on silica gel (packed in toluene and eluted with ethyl acetate) resulted in crude product. The crude product was dissolved in chloroform and washed sequentially with water, 0.5 M NaHCCq and saturated KC1. The chloroform extract was evaporated, and the product was purified on a silica gel column (packed in chloroform and eluted with chloroform/methanol 15:1). Evaporation of fractions and vacuum drying of residue resulted in a colorless thick syrup of (3) (1785 mg, 95%). TLC: Rf = O. 49 (chloroform/methanol 7:1). ΧΗ NMR (500 MHz, [D6]DMSO, 30 °C) δ = 7.826 (t, J= 5.1 Hz, 1H; NHCO), 6.979 (t, J= 5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH2COO), 3.969 (d, J= 5.1 Hz, 2H; COCH2NH) , 3.689 and 3.621 (s, 3H; 00¾) , 3.559 (d, J= 5.9 Hz, 2H; COCH2NHCOO) , 1.380 (s, 9H; CMe3) ppm.
Preparation of {[2-(2-tert-hutoxycarbonylamino-acetylamino) -acetyl]-methoxycarbonylmethyl-amino}-acetic acid
To the stirred solution of { [2-(2-tert-butoxycarbonylamino-acetylamino) -acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2 M aqueous NaOH (23.5 ml) was added. The solution was kept for 5 min at r.t., then acidified with acetic acid (0.6 ml) and evaporated to dryness. Column chromatography of the residue on silica gel (packed in ethyl acetate and eluted with iPrOH/ethyl acetate/water (2:3:1)) resulted in recovered (3) (63 mg, 3.4%) and crude target compound (1320 mg).
The crude target compound was dissolved in methanol/water/pyridine mixture (20:10:1, 30 ml) and passed through an ion-exchange column (Dowex 50X4-400, pyridine form, 5 ml) to remove residual Na cations. The column was washed with the same mixture, eluant evaporated, dissolved in chloroform/benzene mixture (1:1, 50 ml) then evaporated and dried in vacuum to provide a yield of pure (10) was 1250 mg (74%), white solid. TLC: Rf = 0.47 (iPrOH/ethyl acetate/water ( 4:3:1) ) . ΧΗ NMR (500 MHz, [De]DMSO, 30 °C) of mixture of cis- and trans- conformers of N-carboxymethyl-glycine unit c.3:l.
Major conformer: δ = 7.717 (t, J = 5 Hz, 1H; NHCO), 7.024 (t, J = 5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH2COOMe), 3.928 (d, J = 5 Hz, 2H; COCH2NH) , 3.786 (s, 2H; NCH2COOH) , 3.616 (s, 3H; OCH3), 3.563 (d, J = 5.9 Hz, 2H; COCH2NHCOO) , 1.381 (s, 9H; CMe3) ppm.
Minor conformer: δ = 7.766 (t, J — 5 Hz, 1H; NHCO), 7.015 (t, J — 5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH2COOMe), 3.928 (d, J = 5 Hz, 2H; COCH2NH) , 3.858 (s, 2H; NCH2COOH), 3.676 (s, 3H; OCH3), 3.563 (d, J = 5.9 Hz, 2H; COCH2NHCOO), 1.381 (s, 9H; CMe3) ppm.
Preparation of {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid N-oxysuccinimide ester Boc-Gly2(MCMGly) Nos
To an ice-cooled stirred solution of {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid (1200 mg, 3.32 mmol) and N-hydroxysuccinimide (420 mg, 3.65 mmol) in DMF (10 ml) N,N'-dicyclohexylcarbodiimide (754 mg, 3.65 mmol) was added. The mixture was stirred at 0 °C for 30 min, then for 2 h at r.t. The precipitate of N,N'-dicyclohexylurea was filtered off, washed with DMF (5 ml) and the filtrates evaporated to a minimal volume. The residue was agitated with Et20 (50 ml) for 1 h. An ether extract was removed by decantation, and the residue dried in vacuum to yield the target compound (1400 mg, 92%) as a white foam. TLC: Rf = 0.71 (acetone/acetic acid 40:1). XH NMR (500 MHz, [Dg]DMSO, 30 °C), mixture of cis- and trans- conformers of N-carboxymethyl-glycine unit c. 3:2.
Major conformer: δ = 7.896 (t, J = 5.1 Hz, 1H; NHCO) , 6.972 (t, J = 5.9 Hz, 1H; NffCOO), 4.533 (s, 2H; NCH2COON), 4.399 (s, 2H; NCH2COOMe) , 3.997 (d, J= 5.1 Hz, 2H; COCH2NH), 3.695 (s, 3H; OCH3) , 3.566 (d, J = 5.9 Hz, 2H; COCJ72NHCOO) , 1.380 (s, 9H; CMe3) ppm.
Minor conformer: δ = 7.882 (t, J = 5.1 Hz, 1H; NHCO), 6.963 (t, J = 5.9 Hz, 1H; NiTCOO) , 4.924 (s, 2H; NCH2COON) , 4.133 (s, 2H; NCH2COOMe), 4.034 (d, J = 5.1 Hz, 2H; COCJANH) , 3.632 (s, 3H; OCH3) , 3.572 (d, J = 5.9 Hz, 2H; COCJi2NHCOO) , 1.380 (s, 9H; CMe3) ppm.
Preparation of the constructs designated Mai-(CH2) 2CO-CMG (2)-Ad-DOPE and H-CMG (2)-Ad-DOPE
The construct designated H-CMG(2)-Ad-DOPE was prepared from {[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid N-oxysuccinimide ester Boc-Gly2 (MCMGly) Nos according to Scheme III of the publication of Bovin et al (2008) . The construct designated Mal-(CH2)2CO-CMG(2)-Ad-DOPE was prepared according to the first step of Scheme IV of the publication of Bovin et al (2008) . Briefly, the construct designated H-CMG(2)-Ad-DOPE was treated with a 5-fold excess of 3-maleimidopropionic acid oxybenztriazol ester in i-PrOH-water. The maleimide-lipid construct was isolated in 40% yield after gel-permeation chromatography on Sephadex LH-20 (i-PrOH-water, 1:2).
SCHEME A SCHEME B SCHEME C
SCHEME D(a)
SCHEME D(b)
Preparation of NCSeCH2CO-CMG (2) -Ad-DOPE
Attempts to prepare a cyanoselenide-lipid construct via an addition reaction between the maleimide-lipid construct designated Mai-(CH2) 2CO-CMG(2)-Ad-DOPE and potassium selenosulfite (K2SeSo3) [SCHEME A], selenophenol (PhSeH) [SCHEME B] and hydrogen selenide (H2Se) [SCHEME C] were unsuccessful. With hindsight the failure to obtain a stable seleno-Bunte salt according to SCHEME A is at least in part predictable from the disclosure of the chemical behaviour of their sulfur analogues in the publication of Distler (1967). Both the attempted Michael additions of phenylselenide and hydrogen selenide in protic media according to SCHEME B and SCHEME C, respectively, yielded a product with a reduced maleimide double bond, as opposed to the desired selenylsuccinimides. Formation of selenylsuccinimides in quantitative yield has been disclosed in the publication of Numeo et al (1981). However, the disclosed use of anhydrous ether is incompatible with the use of the polyanionic maleimide-lipid construct designated Mai-(CH2) 2CO-CMG(2)-Ad-DOPE.
It was subsequently discovered that the cyanoselenide-lipid construct designated NCSeCH2C0-CMG(2)-Ad-DOPE could be successfully prepared via an activated 2-selenocyanatoacetic acid (NC-Se-CH2COOH). The activated NC-Se-CH2COOH was reacted with the lipid construct H-CMG(2)-Ad-DOPE according to SCHEME D(a) or SCHEME D(b). The prepared construct was stored in the dark under an inert atmosphere. Potassium selenocyanate was selected as the reagent of choice as it could readily be activated as an N-hydroxysuccinimide (NHS) ester according to SCHEME D(a) or (b) or mixed anhydride according to SCHEME D(c). Potassium selenocyanoacetate (NCSeCH2COOK) was synthesized from freshly prepared solutions of potassium selenocyanate (KSeCN) and potassium bromoacetate (BrCH2C00K) according to the procedures disclosed in the publication of Klauss (1970). The synthesized NCSeCH2COOK was stored in a vacuum desiccator over potassium hydroxide (KOH) pellets in the dark prior to activation. For activation the potassium selenocyanoacetate (156 mg, 0.77 mmol) was added in one portion to a solution of N,N,N',N'-tetramethyl-O-(N-succinimidyl)uraniumhexafluorophosphate (HSTU) (IRIS, Germany) (212 mg, 0.59 mmol) in 1 mL DMF while a gentle flow of dry argon via a PTFE capillary was bubbling through. The slurry thus obtained was stirred in this way for 30 minutes during which the initial solid changed to a more dense crystalline precipitate (KPFg) . The reaction mixture was sonicated for 1 to 2 minutes and combined with the construct designated H-CMG(2)-Ad-DOPE (110 mg, 0.06 mmol) dissolved in 1 mL of 20% IPA followed by 100 pL IN KHCO3. A sticky solid (presumably NCSeCH2COOSu) that precipitated immediately, was dissolved by dropwise addition of 30% IPA (circa 1.6 mL) with sonication and the reaction mixture was magnetically stirred for 3 hours at room temperature keeping pH in the range Θ.0 to 8.5 (TLC control: Solvents were evaporated in vacuum and dry residue was triturated with 3 mL of acetonitrile with sonication until fine slurry formed and then transferred into Eppendorf tubes (2 x 2.2 mL), centrifuged and the solids washed 4 times consecutively with neat IPA and MeCN (2 mL of each, brief sonication followed by centrifugation). The wet solids were dissolved in 3.5 mL of 30% IPA-water and lyophilized to constant weight. Ill mg (92%) of the cyanoselenide-lipid construct designated NCSeCH2CO-CMG(2)-Ad-DOPE were obtained as a reddish amorphous powder. Rf-0.5, CHCl3/methanol/water 2:5:1 (v/v); TLC aluminium sheets Silica gel 50 F254 (Merck 1.05554). It is noted that mass spectroscopy did not appear suitable for the characterization of this construct. Only peaks of Se-free fragments could be detected. The 1H NMR spectrum determined for the construct is provided in Figure 1.
Cations as the functional moiety
The polycation lipid construct 9a was prepared and isolated as its trifluoroacetic acid (TFA) salt (SCHEME E). Briefly, desymmetritisation of the polyamine spermine [CAS# 71-44-3] (2) was performed according to a modified version of the method disclosed in the publication of Geall and Blagbrough (2000) employing Boc as the protecting group. It will be recognised that the method is also applicable to the desymmetritisation of other unbranched polyamines such as spermidine [CAS# 124-20-9] (1), tetraethylenepentamine [CAS# 112-57-2] (3); pentaethylenehexamine [CAS# 406716-7] (4) and hexaethyleneheptamine [4403-32-1] (5) . Accordingly, a series of polycation lipid constructs may be accessed according to SCHEME E.
According to SCHEME E the Boc protected, desymmetritised intermediate N1, N4, N9-tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazadodecane (6) is conjugated to the diacylglycerophospholipid 1,2-0-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine [CAS# 4004-05-1] (DOPE) using the homobifunctional crosslinker disuccinimidyl adipate. It will be recognised that other disuccinimidyl compounds may be employed as the homobifunctional crosslinker. These include
The activated lipid (7a) acylates the terminal, primary amino group of N1, N4, N9-tri-tert-butoxycarbonyl)-1,12-diamino-4,9-diazadodecane (6) to provide a lipidated Boc protected polyamine intermediate (8a). Again, it will be recognised that according to Scheme I other diacylglycerophospholipids, such as 1,2-0-distereoyl-sn-glycero-3-phosphatidylethanolamine [CAS# ](DSPE) may be substituted for DOPE.
In the final step of SCHEME A the lipidated polyamine intermediate (8a) is deprotected and the polycation lipid construct (9a) isolated as its trifluoroacetic acid salt.
Materials and methods
Chloroform, dichloroethane, dichloromethane, methanol and toluene were obtained from Chimmed (Russian Federation). Trifluoroacetic acid, triethylamine, di-tert-butyldicarbonate methyl trifluoroacetate were obtained from Merck (Germany). Spermine was obtained from Sigma-Aldrich (USA).
Sephadex LH-20 was obtained from Amersham Biosciences AB (Sweden). Silica gel 60 was obtained from Merck (Germany). Thin layer chromatographic (TLC) analysis was performed on silica gel 60 F254 plates (Merck). Amino containing compounds were detected using ninhydrin reagent. DOPE containing compounds were detected using an aqueous solution of potassium permanganate (KMnCA) or by soaking in 8% (w/v) phosphoric acid in water followed by heating at over 200 °C. 1H NMR spectra were recorded at 30 °C with a Bruker BioSpin GmbH 700 MHz instrument using the signal of the solvent's residual protons as reference ([D]CHCl3, 7.270 ppm; [D2]H20, 4.750 ppm). Mass spectra were recorded with an Agilent ESI-TOF 6224 LC/MS spectrometer.
Preparation of Boc3Spm (6)
To a stirred solution of spermine (2) (1 equivalent, 1.34 g, 6.6 mmol) in methanol (90 mL) at -80 °C under nitrogen, a solution of methyl trifluoroacetate (1.1 equivalents, 0.730 mL, 7.26 mmol) in methanol (1.5 mL) was added drop-wise over a period of 30 min. Stirring was continued at -80°C for a further period of 30 min and then the temperature increased to 0 °C.
The reaction afforded predominantly the mono-trifluoroacetamide. Without isolation, the remaining amino functional groups were quantitatively protected by drop-wise addition of an excess of di-tert-butyldicarbonate (4 equivalents, 5.76 g, 26.4 mmol) in methanol over a period of 3 min. The reaction was then warmed to 25 °C and stirred for a further 15 hr to afford the fully protected spermine (Rf 0.33 (95:5 (v/v) CHCl3-i-PrOH)). The trifluoroacetate protecting group was then removed in situ by increasing the pH of the solution to greater than 11 pH units with concentrated aqueous ammonia (cone. aq. NH3) and then stirred at 25 °C for a period of 15 hr. The solution was concentrated in vacuo and the residue purified over silica gel (95:5:1 to 90:10:1 (v/v/v) CHCl3-MeOH-conc. aq. NH3) to afford the title compound (6) as a colourless homogeneous oil (1.5 g, 45%), Rf 0.32 (83:16:1 (v/v/v) CHCl3-MeOH-conc. aq. NH3) . MS, m/z: found 502.3725 (M++l), C25H50N4O6 required M+ 501.3652. 1H-NMR (700 MHz, CDCI3, 303 °K) , δ, ppm: 3.4 (m, 2H, 1-CH2) , 3.05-3.30 (m, 8H, 3,4,7,8-CH2) , 3.01(m, 2H, 10-CH2), 2.03 (m, 2H, 9-CH2), 1.67 (m, 2H, 2-CH2), 1.50 (m, 4H, 5,6-0¾), 1.44, 1.45, 1.46 (3 s, overlapping, 27 H, 3 0-0(0¾^).
Preparation of SuO-Ad-DOPE (7a) and SuO-Ad-DSPE (7b)
To a solution of disuccinimidyl adipate (70 mg, 205 μτηοΐ) in dry N,N-dimethylformamide (1.5 ml) were added DOPE or DSPE (40pmol) in chloroform (1.5 ml) followed by triethylamine (7 μΐ). The mixture was kept for 2 h at room temperature, then neutralized with acetic acid and partially concentrated in vacuo. Column chromatography (Sephadex LH-20, 1:1 (v/v) chloroform-methanol, 0.2% (w/v) aqueous acetic acid) of the residue yielded
SuO-Ad-DOPE (7a)(37 mg, 95%) as a colourless syrup. TLC (6:3:0.5 (v/v/v) chloroform-methanol-water) Rf 0.5 (SuO-Ad-DOPE (7a)) and Rf 0.55 (SuO-Ad-DOPE (7b)). ΧΗ NMR (2:1 (v/v) CDCI3/CD3OD) δ:
SuO-Ad-DOPE (7a) - 5.5 (m, 4H, 2x(-CH=CH-), 5.39 (m, 1H, -0CH2-CH0-CH20-), 4.58 (dd, 1H, 3=3.61, J=11.98, -CCOOHCH-CHO-CH2O-) , 4.34 (dd, 1H, J=6.61, J= 11.9 8, -CCOOHCH-CHO-CH2O-) , 4.26 (m, 2H, PO-CH2-CH2-NH2) , 4.18 (m, 2H, -CH2-
OP), 3,62 (m, 2H, PO-CH2-CH2-NH2 ) , 3.00 (s, 4H, ONSuc) , 2.8 (m, 2H, -CH2-CO (Ad), 2.50 (m, 4H, 2x (-CH2-CO) , 2.42 (m, 2H, -CH2-CO (Ad), 2.17 (m, 8H, 2x(- CH2-CH=CH-CH2-) , 1.93 (m, 4H, COCH2CH2CH2CH2CO) , 1.78 (m, 4H, 2x (COCH2CH2-) , 1,43, 1.47 (2 bs, 40H, 20 CH2), 1.04 (m, 6H, 2 CH3) .
SuO-Ad-DSPE (7b) - 5.39 (m, 1H, -0CH2-CH0-CH20-) , 4.53 (dd, 1H, J=3.42, J=11.9 8, -CCOOHCH-CHO-CH2O-) , 4.33 (dd, 1H, 3=6.61, J=11.98, -CCOOHCH-CHO-CH20-), 4.23 (m, 2H, PO-CH^-CH2-NH2) , 4.15 (m, 2H, -CH2-OP) , 3,61 (m, 2H, PO-CH2-CH2-NH2) , 3.00 (s, 4H, ONSuc), 2.81 (m, 2H, -CH2-CO (Ad), 2.48 (m, 4H, 2x(- CH2-CO) , 2.42 (m, 2H, -CH2-CO (Ad), 1.93 (m, 4H, COCH2CH2CH2CH2CO) , 1.78 (m, 4H, 2x(COCH2CH2-) , 1,43, 1.47 (2 bs, 40H, 20 CH2), 1.04 (m, 6H, 2 CH3) .
Preparation of Boc3Spm-Ad-DOPE (8a)
To a stirred solution of Boc3Spm (6) (552 mg, 1.1 mmol) in dichloroethane (25 ml) was added trimethylamine (1 ml, 7.2 mmol) followed by a solution of SuO-Ad-DOPE (1066 mg, 1.1 mmol) in dichloroethane (25 ml). The reaction mixture was stirred for a period of 2 hr and then the solvent was removed under reduced pressure at 37 °C. The crude product was purified by chromatography on silica gel by elution with 97:3 to 85:15 (v/v) CHCl3-MeOH to afford the title compound (8a) (1.16 g, 78%) as a viscous oil. TLC (10:6:0.8 (v/v/v) CH2Cl2-Et0H-H20) Rf 0.36. bH NMR (700 MHz, CDCI3/CD3OD 1:1, 10 mg/mL, 303 °K) δ, ppm: 5.34 (m, 4H; 2 CH=CH), 5.19 (m, 1H; 0CH2CHCH20), 4.37 (dd, Jgem~ll.l Hz, 1H, POCH2-CH-CHa-O(CO)), 4.13 (dd, J-7.2 Hz, 1H, P0CH2-CH-CHb-0(CO)), 3.94 (m, 4H), 3.48(m, 2H), 3.05-3.30 (m, 12H, 1,3,4,7,8,10-CH2) , 2.71 (m, 2H) , 2.20-2.42 (m, 8H) , 1.98-2.04 (m, 8H), 1.64 (m, 8H,), 1.58 (m, 4H), 1.49 (m, 4H, 5,6-CH2), 1.44, 1.45, 1.46 (3s, 27H, 3 0-C(CH3)3), 1.22-1.37 (m, 40H, 20 CH2) , 0.88 and 0.89 (2d, J«7 Hz, 6H, 2 CH3) .
Preparation of Spm-Ad-DOPE (9a)
To a stirred solution of 8a (1.16 g, 0.85 mmol) in CHC13 (10 ml) at 25 °C TFA (5 ml, 95%) was added. After a period of 20 min the solution was concentrated in vacuo at 35 °C and the residue was co-evaporated with toluene (5 times 10 mL) to remove trace amounts of TFA. To remove any low molecular weight impurities the residue was dissolved in 1:1 (v/v) CHCl3-MeOH (2 mL) and passed in two portions through a Sephadex LH-20 column (volume 330 mL, eluent 1:1 (v/v) CHCl3-MeOH). Fractions containing pure 9a (di-TFA salt) were combined and evaporated to dryness and the residue dissolved in water (-100 mL) and freeze-dried. A yield of was 975 mg (89%) was obtained. MS, m/z: found 1056.8063 (M++l), C57H110N5O10P required M+ 1055.779. bH NMR (700 MHz, 1:1 (v/v) CDC13-CD30D, 10 mg/mL, 303°K) δ, ppm: 5.51 (m, 4H; 2 CH=CH), 5.42 (m, 1H; 0CH2CHCH20), 4.6 (dd, Jgem=12.1 Hz, J=2.81 Hz, 1H, P0CH2-CH-CHa-0 (CO) ) , 4.34 (dd, J=7 . 09 Hz, 1H, P0CH2-CH-CHb-0(CO)), 4.14 (m, 2H, POCH2CH2N) , 4.06 (m, 2H, POCH2-CH-CH2) , 3.59 (m, 2H, OCH2CH2N), 3.49 (m, 2H, 1-CH2), 3.11-3.28 (m, 10H, 3,4,7,8,10-CH2) , 2.42 and 2.51 (2m, 8H, 4 COCH2), 2.26 (m, 2H, 2-CHz) , 2.19 (m, 8H, 2 CH2CH=CHCH2) , 2.07 (m, 2H, 9-CH2) , 1.99 (m, 4H, 5,6-CH2) , 1.79 (m, 8H, 4 COCH2CH2) , 1.40-1.54 (m, 40H, 20 CH2) , 1.05 and 1.06 (21, J*7 Hz, 6H, 2 CH3) .
SCHEME E
Surface treatment - antimicrobial
The ability of the polycation-lipid construct designated Spm-Ad-DOPE (9a) to prevent the growth of bacteria on the surface of stainless steel was evaluated. A dispersion of the construct was prepared at a concentration of 1 mg/mL in sterile deionised water. (It is noted that attempts to disperse the construct in saline resulted in precipitation of the construct.) A volume of 100 pL of the dispersion was dispensed onto the surface of a 1 x 1 cm stainless steel (SS 304) square. A control was prepared by dispensing the same volume of sterile deionised water onto the surface of a second stainless steel square. Both samples (test and control) were then dried at 60°C for a period of two hours . The samples were stored at room temperature prior to use. A volume of 1 mL of an actively growing (log phase) culture of
Escherichia coli (ATCC 25922) in 21 g/L Mueller-Hinton broth (MHB) was serially diluted (10-6) to provide 8 to 10 colony forming units (CFUs) per 100 pL . Individual samples of the stainless steel squares were placed in each well of a sterile 12-well culture plate and 100 mL of the serially diluted culture dispensed onto the surface of each sample. The culture was allowed to contact the surface for a period of 20 minutes at room temperature before washing each sample once with phosphate buffered saline (PBS) to remove nonadherent cells of the bacterium. Each washed sample was then immersed in a volume of 10 mL of MHB and incubated overnight at 37°C. Following overnight incubation each sample was washed as before and immersed in a volume of 9 mL of MHB. Alternate vortexing and sonicating was employed to remove bacteria from the sample surface. A volume of a serial dilution (10-4) of the resulting broth was then spread on blood agar plates, incubated at 37°C overnight and colonies counted. Cell densities of the overnight cultures were calculated and are presented in Table 1.
Table 1. Growth of overnight cultures of Escherichia coli (ATCC 25922) following contact with surface treated (Test) and untreated (Control) of lxl cm stainless steel samples.
The tabulated results indicate a biocidal action of the samples treated with the polycation-lipid construct designated Spm-Ad-DOPE (9a).
The ability of the following constructs to prevent the growth of clinical isolates to Staphylococcus aureus and Staphylococcus epidermidis to grow on the surface of stainless steel (SS 316) was evaluated: • The construct designated NCSeCH2CO-CMG(2)-Ad-DOPE as described in the specification accompanying international application no. PCT/NZ2015/05 0181 [publ. no. WO 2 016/072 8 53]; • The construct designated Spm-Ad-DOPE (9a); and • The construct designated HA-gar-Ad-DOPE as described in the specification accompanying international application no. PCT/NZ2006/000245 [publ. no. WO 2007/035116],
Frozen stocks of the Staphylococcus sp. inocula were thawed at 37°C for 10 minutes and vortexed before plating on blood agar and incubating at 37°C for 18 hours. Single colonies were used to inoculate a volume of 10 mL of
Mueller-Hinton Broth (MHB) and incubated at 37°C for 18 hours in a shaking incubator (200 rpm). A volume of 100 pL of the actively growing culture was then used to inoculate a volume of 100 mL of MHB and incubated at 37°C for 6½ hours. The turbid culture suspension (OD600 = 1.596) was serially diluted to 10~8 in eight volumes of 9 mL of MHB. A volume of 100 pL of each dilution step was spread on plate count agar (PCA) plates and incubated at 37°C for 18 hours to confirm viable cell numbers. The dilution to 10~7 was used in the following steps .
Volumes of 100 pL of dispersions in water of each construct were dispensed onto the surface of stainless steel squares (5 replicates); • 2 mg/mL NCSeCH2CO-CMG(2)-Ad-DOPE; • 1 mg/mL Spm-Ad-DOPE (9a); and • 1 mg/mL HA-gar-Ad-DOPE.
The stainless steel squares were then dried at 60°C for 30 minutes and kept at room temperature before use. Each one of the treated stainless steel squares was then placed in an individual well of a sterile multi-well plate and volumes of 100 pL of the dilution to 10~7 of actively growing Staphylococcus sp. isolate dispensed into each well. The plate was then incubated at 37°C for 18 hours with shaking (200 rpm). The stainless steel squares were each then removed from their respective wells and washed 3 times in volumes of 1 mL of sterile Maximum Recovery Diluent (MRD) and then placed in a volume of 9 mL of MRD and serially diluted to 10~4 as before. For each dilution step a volume of 100 pL was spread on a PCA plate, allowed to dry and then incubated at 37°C for 18 hours and colonies counted. The surface of the stainless steel squares was also examined by SEM following fixing in 2.5% glutaraldehyde (4°C overnight), dehydration with increasing concentrations of ethanol, drying and sputter-coating with platinum for 60 seconds and imaging at 5.0 kV at 4,500 x magnification. Ten fields selected at random were counted for each sample. Electron micrographs of randomly selected fields are provided in Figure 8.
The susceptibility of clinical isolates of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa to the constructs designated Spm-Ad-DOPE (9a) and NCSeCH2CO-CMG(2)-Ad-DOPE was determined by broth microdilution. Actively growing cultures of each clinical isolate were prepared as described above to obtain cultures with the following turbidities (ΟΌβοο) : • Staphylococcus aureus 1.371 • Escherichia coli 1.300 • Pseudomonas aeruginosa 0.906
Serial dilutions of each culture to 1CV6 were prepared and used as inocula. Volumes of 50 pL of each dilution was spread on PCA plates and incubated at 37°C for 18 hours. A two-fold dilution series for each of the two constructs, spermine (2) and selenous acid (H2Se03) were prepared in sterile 96-well plates . Volumes of 50 pL of inocula were dispensed into a well containing a volume of 50 pL of the serially diluted construct, spermine (2) or selenous acid (EhSeCp) . Following gentle mixing the plates were incubated in the dark at 37°C for 18 hours (200 rpm). Minimum inhibitory concentrations (MIC) were thereby observed and minimum bactericidal concentrations (MBC) determined by plating aliquots from each well. Both constructs were observed to be more effective bacteriostatic (Table 2) or bactericidal (Table 3) agents then either of their functional moieties (spermine or selenous acid) alone.
Table 2. Minimum inhibitory concentrations (MICs) as determined by broth microdilution. MBC reading in μΜ
Clinical isolate
Table 3. Minimum bactericidal concentrations (MBCs) as determined by broth microdilution and plating.
Surface treatment - micro-dimensioned particle (biotic origin) adherence A stock solution of the construct designated Spm-Ad-DOPE (9a) was prepared in methanol at a concentration of 10 mg/mL. The stock solution was diluted to a concentration of 250 pg/mL in methanol and 25 pL volumes of the diluted stock solutions dispensed into each of the round bottomed wells of a multi-well microplate (Corning Inc.). The plates were allowed to dry before washing the wells 6 times with deionized water. Control wells were similarly treated using either methanol alone (blank) or substituting the construct designated Biotin-CMG(2)-Ad-DOPE as described in the specification accompanying international application no. PCT/NZ2008/000266 [publ. no. WO 2009/048343]).
Red blood cells (RBCs; group 0, up to 2 weeks old) were washed and resuspended at a concentration of 1% packed cell volume (pcv) in phosphate-buffered saline (PBS). A 50 pL volume of the suspension of RBCs was dispensed into each of the wells and incubated for 1 hour at room temperature before washing 6 times with PBS. The RBCs were fixed by adding a 50 pL volume of a solution of glutaraldehyde in PBS at a concentration of 2.5% (w/v) and incubating for 10 minutes before washing each well with water and allowing to dry. The RBCs were lysed by adding a 50 pL volume of deionized water and incubating for 10 minutes before discarding the water and allowing to dry.
For scanning electron microscopy (SEM) the bottom of each well was cut from the plate and the treated surface sputter coated with platinum prior to imaging. The images obtained at increasing magnification for well surfaces treated according to the methods described and those obtained for commercially available plates (Capture-R™ Ready-Id, Capture-R™ Ready-Screen and CT-6; Immucor Inc.) are provided in Figure 10.
Adherence of RBCs to well surfaces treated according to the method described was clearly evident. Investigations were performed to determine if the adherence could be attributable to the use of the construct designated Spm-Ad-DOPE (9a) or the polycation spermine (2) alone. A stock solution of spermine (2) was prepared at a concentration of 10 mg/mL in methanol. The spermine stock solution was diluted to a concentration of 0.2 mg/mL in either methanol or water. Volumes of 200 pL of diluted stock solution (approximately 950 μΜ) of the construct designated Spm-Ad-DOPE (9a), spermine (2) in methanol or spermine in water were added to each of the first wells of a microplate. A two-fold serial dilution from each of the first wells was then performed. The microplate was then dried under vacuum before washing each well of the microplate with deionized water by immersing and discarding the wash water 4 times. After drying, 50 pL volumes of a suspension of RBCs at a concentration of 1% pcv in either 10 mM Tris/0.25 M sucrose (SucT) or PBS were dispensed into each well. (Aggregation of RBCs was observed in wells where the construct designated Spm-Ad-DOPE (9a) had been added at a concentration of 29 μΜ or greater.) The plate was incubated for one hour at room temperature before washing the wells 6 times with PBS.
Following drying the plate was inverted and the base of the wells examined by light microscopy (lOOx magnification) for the presence of a uniform monolayer of RBCs. Photomicrographs obtained for wells treated with a solution of either the construct designated Spm-Ad-DOPE (9a) or spermine (2) alone are presented in Figure 11. A uniform monolayer of cells was observed in wells treated with the construct designated Spm-Ad-DOPE (9a) at concentrations of 14 to 237 μΜ (0.015 to 0.25 mg/mL) in either of the two buffers (SucT or PBS) used. Any attachment of cells when spermine (2) alone was used was not uniform or reproducible. No cells were observed to adhere to the surface in wells containing spermine (2) alone in water.
Micro-dimensioned particle (abiotic origin) adherence A volume of 100 pL of a solution of 0.05% (w/v) bromophenol blue and 50 pM of the construct designated KODE-spm in water was dispensed and spread across the surface of a strip of laminated nylon mesh. The strip was allowed to dry for one hour at room temperature before being exposed to particulates released from either smoking cigarettes or a wood burner using an artificial syringe as a "puffer" (exposure for about 10 minutes). The exposed strips were stored in a sealed polythene bag before being examined by scanning electron microscopy (SEM) (Figure 12).
Although the invention has been described with reference to embodiments or examples it should be appreciated that variations and modifications may be made to these embodiments or examples without departing from the scope of the invention. Where known equivalents exist to specific elements, features or integers, such equivalents are incorporated as if specifically referred to in this specification. In particular, variations and modifications to the embodiments or examples that include elements, features or integers disclosed in and selected from the referenced publications are within the scope of the invention unless specifically disclaimed. The advantages provided by the invention and discussed in the description may be provided in the alternative or in combination in these different embodiments of the invention.
INCORPORATION BY REFERENCE
The application accompanied by this specification claims the benefit of the priority dates established by the filing of Australian application no. 2015901844 (filed 20 May 2015) and international application no. PCT/NZ2015/050181 (filed 3 November 2015) . The descriptions provided in the specifications accompanying each of these applications, and those of the specifications accompanying international application nos. PCT/NZ2006/000245 [publ. no. WO 2007/035116] and PCT/NZ2008/000266 [publ. no WO 2009/048343], are incorporated here.
INDUSTRIAL APPLICABILITY
The method of surface treating surgical treatments is performed ex vivo using synthetic, water dispersible polycation-lipid constructs.
PUBLICATIONS
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Gunn (2007) Organotellurium and Selenium-Based Antimicrobial Antimicrobial [sic] Formulations and Articles international PCT application no. PCT/US2007/064333 (publ. no. WO 2007/109633 A2)
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Jeney and Zsolnai (1959) Bacteriostatic action of organic selenocyanates Naturwissenschaften, 46, 231
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Public Access Author Manuscript Mater Sci Eng C Mater Biol Appl 33(4) : 20592069
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Claims (6)
- CLAIMS [1] A polycation-lipid construct of the structure:where X is -CH2- and n is the integer 3, 4 or 5; Ri and R2 are independently selected from the group consisting of C14-20 acyl groups that are unbranched and saturated or mono-unsaturated; and R3 is an N1-acylated polyamine.
- [2] The construct of claim 1 where R3 is of the structure:; or
- [3] A polycation-lipid construct of the structure:designated Spm-Ad-DOPE.
- [4] A selenide-lipid construct of the structure:where : m is the integer 1,2,3 or 4; preferably the integer 1, 2 or 4; most preferably the integer 2; n is the integer 3, 4 or 5; most preferably the integer 4; p is the integer 1, 2 or 3; most preferably the integer 2; q is the integer 1, 2 or 3; most preferably the integer 1; M is a monovalent substituent; preferably the monovalent substituent CH3 or H; most preferably the monovalent substituent H; M' is a monovalent cation or substituent; preferably the monovalent cation H+, K+ or Na+; most preferably the monovalent cation H+; and Ri and R2 are independently an aliphatic C14-20 acyl, aliphatic C14-20 alkenyl or aliphatic C14-20 alkyl substituent; preferably a substituent selected from the group consisting of myristyl, palmityl, stearyl, arachidyl, palmitoleoyl, petroselenyl, oleoyl, elaidyl, vaccenyl and gondoyl; most preferably the aliphatic Ci8 alkenyl substituent oleoyl.
- [5] A surface treatment preparation comprising a dispersion in water of a construct of any one of claims 1 to 4.
- [6] A surface treatment method comprising the step of contacting a surface with the preparation of claim 5.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2015901844A AU2015901844A0 (en) | 2015-05-20 | Surface treatment | |
| AU2015901844 | 2015-05-20 | ||
| AUPCT/NZ2015/050181 | 2015-11-03 | ||
| PCT/NZ2015/050181 WO2016072863A1 (en) | 2014-11-03 | 2015-11-03 | Antimicrobial surface treatment |
| PCT/IB2016/052735 WO2016185331A1 (en) | 2015-05-20 | 2016-05-12 | Surface treatments |
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| AU2016262958A1 true AU2016262958A1 (en) | 2018-01-18 |
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| AU2016262958A Abandoned AU2016262958A1 (en) | 2015-05-20 | 2016-05-12 | Surface treatments |
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| CN (1) | CN108137627A (en) |
| AU (1) | AU2016262958A1 (en) |
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| GB2463584B (en) * | 2007-04-27 | 2012-02-01 | Kode Biotech Ltd | Carbohydrate-lipid constructs and their use in preventing or treating viral infection |
| NZ591047A (en) * | 2011-02-09 | 2013-11-29 | Vladimirovich Bovin Nicolai | In vivo methods of monitoring biodistribution |
| AU2013200205B2 (en) * | 2011-08-31 | 2014-10-30 | Nikolai Vladimirovich Bovin | Facile laboratory method for localising biomolecules to the surface of cells and viruses |
| EP2912045A4 (en) * | 2012-10-29 | 2016-07-13 | Molecular Transfer Inc | Polycationic methyl phospholipids for improved delivery of nucleic acids to eukaryotic cells |
-
2016
- 2016-05-12 GB GB1721384.4A patent/GB2564917A/en not_active Withdrawn
- 2016-05-12 AU AU2016262958A patent/AU2016262958A1/en not_active Abandoned
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| GB2564917A (en) | 2019-01-30 |
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