US20230149567A1 - Mechanical opening of lipid bilayers by molecular nanomachines - Google Patents
Mechanical opening of lipid bilayers by molecular nanomachines Download PDFInfo
- Publication number
- US20230149567A1 US20230149567A1 US18/149,934 US202318149934A US2023149567A1 US 20230149567 A1 US20230149567 A1 US 20230149567A1 US 202318149934 A US202318149934 A US 202318149934A US 2023149567 A1 US2023149567 A1 US 2023149567A1
- Authority
- US
- United States
- Prior art keywords
- cells
- molecule
- groups
- molecules
- cell
- 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
- 239000000232 Lipid Bilayer Substances 0.000 title claims abstract description 128
- 239000013543 active substance Substances 0.000 claims abstract description 38
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 36
- 229940079593 drug Drugs 0.000 claims abstract description 14
- 239000003814 drug Substances 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims description 44
- 239000003795 chemical substances by application Substances 0.000 claims description 37
- 239000000975 dye Substances 0.000 claims description 26
- 230000008685 targeting Effects 0.000 claims description 25
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 21
- 239000002904 solvent Substances 0.000 claims description 19
- -1 fluorophores Substances 0.000 claims description 12
- 150000004676 glycans Chemical class 0.000 claims description 12
- 239000005017 polysaccharide Substances 0.000 claims description 12
- 229920001282 polysaccharide Polymers 0.000 claims description 12
- 108020004414 DNA Proteins 0.000 claims description 10
- 125000003118 aryl group Chemical group 0.000 claims description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 8
- 150000001720 carbohydrates Chemical class 0.000 claims description 8
- 235000014633 carbohydrates Nutrition 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 108020004459 Small interfering RNA Proteins 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 150000001298 alcohols Chemical group 0.000 claims description 6
- 150000001345 alkine derivatives Chemical group 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 150000001335 aliphatic alkanes Chemical group 0.000 claims description 5
- 150000001336 alkenes Chemical group 0.000 claims description 5
- 125000003368 amide group Chemical group 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 5
- 229910052739 hydrogen Inorganic materials 0.000 claims description 5
- 239000001257 hydrogen Substances 0.000 claims description 5
- 150000002825 nitriles Chemical group 0.000 claims description 5
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 150000007942 carboxylates Chemical class 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 150000007513 acids Chemical class 0.000 claims description 3
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 150000002170 ethers Chemical group 0.000 claims description 3
- 150000002334 glycols Chemical class 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- 150000002367 halogens Chemical group 0.000 claims description 3
- 125000000468 ketone group Chemical group 0.000 claims description 3
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 3
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 150000003871 sulfonates Chemical group 0.000 claims description 3
- 150000003467 sulfuric acid derivatives Chemical group 0.000 claims description 3
- 108091023037 Aptamer Proteins 0.000 claims description 2
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 2
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 0.000 claims description 2
- 150000001413 amino acids Chemical class 0.000 claims description 2
- 239000002872 contrast media Substances 0.000 claims description 2
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 claims description 2
- 229910052733 gallium Inorganic materials 0.000 claims description 2
- 229920000570 polyether Polymers 0.000 claims description 2
- 230000002285 radioactive effect Effects 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 229910052716 thallium Inorganic materials 0.000 claims description 2
- BKVIYDNLLOSFOA-UHFFFAOYSA-N thallium Chemical compound [Tl] BKVIYDNLLOSFOA-UHFFFAOYSA-N 0.000 claims description 2
- 150000002431 hydrogen Chemical group 0.000 claims 4
- 210000004027 cell Anatomy 0.000 abstract description 318
- 238000000034 method Methods 0.000 abstract description 92
- 239000000463 material Substances 0.000 abstract description 28
- 210000000170 cell membrane Anatomy 0.000 abstract description 27
- 239000011148 porous material Substances 0.000 abstract description 22
- 238000001727 in vivo Methods 0.000 abstract description 13
- 230000015572 biosynthetic process Effects 0.000 abstract description 12
- 238000000338 in vitro Methods 0.000 abstract description 11
- 230000004044 response Effects 0.000 abstract description 11
- 230000017074 necrotic cell death Effects 0.000 description 67
- 239000012528 membrane Substances 0.000 description 55
- 238000002474 experimental method Methods 0.000 description 51
- 238000005286 illumination Methods 0.000 description 27
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 27
- 238000005406 washing Methods 0.000 description 27
- 238000003384 imaging method Methods 0.000 description 25
- 230000009471 action Effects 0.000 description 24
- 230000033001 locomotion Effects 0.000 description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 23
- 238000011534 incubation Methods 0.000 description 22
- 230000000694 effects Effects 0.000 description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 18
- 230000005540 biological transmission Effects 0.000 description 17
- 238000000386 microscopy Methods 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 15
- 230000001413 cellular effect Effects 0.000 description 15
- 230000005284 excitation Effects 0.000 description 15
- 230000002438 mitochondrial effect Effects 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- 230000036962 time dependent Effects 0.000 description 14
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 13
- 206010028980 Neoplasm Diseases 0.000 description 12
- 230000004913 activation Effects 0.000 description 12
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 210000003491 skin Anatomy 0.000 description 12
- 238000011068 loading method Methods 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- 230000004660 morphological change Effects 0.000 description 11
- 238000007619 statistical method Methods 0.000 description 11
- 230000006907 apoptotic process Effects 0.000 description 9
- 230000030833 cell death Effects 0.000 description 9
- 210000000056 organ Anatomy 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 230000032258 transport Effects 0.000 description 9
- 108091006146 Channels Proteins 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 229940126214 compound 3 Drugs 0.000 description 8
- 238000002073 fluorescence micrograph Methods 0.000 description 8
- 150000002632 lipids Chemical class 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 201000011510 cancer Diseases 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 210000003463 organelle Anatomy 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- ONBQEOIKXPHGMB-VBSBHUPXSA-N 1-[2-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4,6-dihydroxyphenyl]-3-(4-hydroxyphenyl)propan-1-one Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(O)=C1C(=O)CCC1=CC=C(O)C=C1 ONBQEOIKXPHGMB-VBSBHUPXSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 210000001789 adipocyte Anatomy 0.000 description 6
- 239000006143 cell culture medium Substances 0.000 description 6
- 229940125773 compound 10 Drugs 0.000 description 6
- 229940126142 compound 16 Drugs 0.000 description 6
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
- 230000008823 permeabilization Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000010859 live-cell imaging Methods 0.000 description 5
- 230000004807 localization Effects 0.000 description 5
- 210000004940 nucleus Anatomy 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 206010060862 Prostate cancer Diseases 0.000 description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 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 4
- 230000005684 electric field Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000012216 imaging agent Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000004417 polycarbonate Substances 0.000 description 4
- 238000011533 pre-incubation Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000000527 sonication Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- MUSGYEMSJUFFHT-UWABRSFTSA-N 2-[(4R,7S,10S,13S,19S,22S,25S,28S,31S,34R)-34-[[(2S,3S)-2-[[(2R)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-4-[[(2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl]-methylcarbamoyl]-25-(3-amino-3-oxopropyl)-7-(3-carbamimidamidopropyl)-10-(1H-imidazol-5-ylmethyl)-19-(1H-indol-3-ylmethyl)-13,17-dimethyl-28-[(1-methylindol-3-yl)methyl]-6,9,12,15,18,21,24,27,30,33-decaoxo-31-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decazacyclopentatriacont-22-yl]acetic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](N)Cc1ccc(O)cc1)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](Cc2cnc[nH]2)NC(=O)[C@H](C)NC(=O)CN(C)C(=O)[C@H](Cc2c[nH]c3ccccc23)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc2cn(C)c3ccccc23)NC(=O)[C@@H](NC1=O)C(C)C)C(=O)N(C)[C@@H]([C@@H](C)CC)C(N)=O MUSGYEMSJUFFHT-UWABRSFTSA-N 0.000 description 3
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 238000012935 Averaging Methods 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 208000003445 Mouth Neoplasms Diseases 0.000 description 3
- 208000000453 Skin Neoplasms Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000862 absorption spectrum Methods 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 238000003782 apoptosis assay Methods 0.000 description 3
- 230000002457 bidirectional effect Effects 0.000 description 3
- 238000004422 calculation algorithm Methods 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 230000007541 cellular toxicity Effects 0.000 description 3
- 230000001427 coherent effect Effects 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940125898 compound 5 Drugs 0.000 description 3
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- FHIVAFMUCKRCQO-UHFFFAOYSA-N diazinon Chemical compound CCOP(=S)(OCC)OC1=CC(C)=NC(C(C)C)=N1 FHIVAFMUCKRCQO-UHFFFAOYSA-N 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 238000007654 immersion Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000005522 programmed cell death Effects 0.000 description 3
- 235000010378 sodium ascorbate Nutrition 0.000 description 3
- 229960005055 sodium ascorbate Drugs 0.000 description 3
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 230000001052 transient effect Effects 0.000 description 3
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 3
- 208000013139 vaginal neoplasm Diseases 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- WTBFLCSPLLEDEM-JIDRGYQWSA-N 1,2-dioleoyl-sn-glycero-3-phospho-L-serine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC WTBFLCSPLLEDEM-JIDRGYQWSA-N 0.000 description 2
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- CSDQQAQKBAQLLE-UHFFFAOYSA-N 4-(4-chlorophenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine Chemical compound C1=CC(Cl)=CC=C1C1C(C=CS2)=C2CCN1 CSDQQAQKBAQLLE-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108090000301 Membrane transport proteins Proteins 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 230000032912 absorption of UV light Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- WEVYAHXRMPXWCK-FIBGUPNXSA-N acetonitrile-d3 Chemical compound [2H]C([2H])([2H])C#N WEVYAHXRMPXWCK-FIBGUPNXSA-N 0.000 description 2
- 230000010933 acylation Effects 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000007697 cis-trans-isomerization reaction Methods 0.000 description 2
- 238000010226 confocal imaging Methods 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000003111 delayed effect Effects 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000002500 effect on skin Effects 0.000 description 2
- 230000005672 electromagnetic field Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000003284 homeostatic effect Effects 0.000 description 2
- 238000009830 intercalation Methods 0.000 description 2
- 230000002687 intercalation Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 201000001441 melanoma Diseases 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- MLBYLEUJXUBIJJ-UHFFFAOYSA-N pent-4-ynoic acid Chemical compound OC(=O)CCC#C MLBYLEUJXUBIJJ-UHFFFAOYSA-N 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 2
- 238000006862 quantum yield reaction Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 201000000849 skin cancer Diseases 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 2
- IHUKVJKKTBLTEE-QMMMGPOBSA-N (2s)-2-acetamido-5-[[amino-(methylcarbamoylamino)methylidene]amino]-n-methylpentanamide Chemical group CNC(=O)NC(N)=NCCC[C@H](NC(C)=O)C(=O)NC IHUKVJKKTBLTEE-QMMMGPOBSA-N 0.000 description 1
- HZHXMUPSBUKRBW-FXQIFTODSA-N (4s)-4-[[2-[[(2s)-2-amino-3-carboxypropanoyl]amino]acetyl]amino]-5-[[(1s)-1-carboxyethyl]amino]-5-oxopentanoic acid Chemical group 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 description 1
- MWRBNPKJOOWZPW-NYVOMTAGSA-N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-NYVOMTAGSA-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
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 125000004070 6 membered heterocyclic group Chemical group 0.000 description 1
- JDDWRLPTKIOUOF-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-[[4-[2-[bis(4-methylphenyl)methylamino]-2-oxoethoxy]phenyl]-(2,4-dimethoxyphenyl)methyl]carbamate Chemical compound COC1=CC(OC)=CC=C1C(C=1C=CC(OCC(=O)NC(C=2C=CC(C)=CC=2)C=2C=CC(C)=CC=2)=CC=1)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 JDDWRLPTKIOUOF-UHFFFAOYSA-N 0.000 description 1
- 108700023418 Amidases Proteins 0.000 description 1
- 102000016614 Autophagy-Related Protein 5 Human genes 0.000 description 1
- 108010092776 Autophagy-Related Protein 5 Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N Cyclohexanecarboxylic acid Natural products OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 108700041152 Endoplasmic Reticulum Chaperone BiP Proteins 0.000 description 1
- 102100021451 Endoplasmic reticulum chaperone BiP Human genes 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 108090000862 Ion Channels Proteins 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 206010034960 Photophobia Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PCBOWMZAEDDKNH-HOTGVXAUSA-N [4-(trifluoromethoxy)phenyl]methyl (3as,6as)-2-(3-fluoro-4-sulfamoylbenzoyl)-1,3,3a,4,6,6a-hexahydropyrrolo[3,4-c]pyrrole-5-carboxylate Chemical compound C1=C(F)C(S(=O)(=O)N)=CC=C1C(=O)N1C[C@H]2CN(C(=O)OCC=3C=CC(OC(F)(F)F)=CC=3)C[C@@H]2C1 PCBOWMZAEDDKNH-HOTGVXAUSA-N 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000000746 allylic group Chemical group 0.000 description 1
- AFVLVVWMAFSXCK-VMPITWQZSA-N alpha-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(\C#N)=C\C1=CC=C(O)C=C1 AFVLVVWMAFSXCK-VMPITWQZSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 125000005228 aryl sulfonate group Chemical group 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 238000010461 azide-alkyne cycloaddition reaction Methods 0.000 description 1
- DMLAVOWQYNRWNQ-UHFFFAOYSA-N azobenzene Chemical compound C1=CC=CC=C1N=NC1=CC=CC=C1 DMLAVOWQYNRWNQ-UHFFFAOYSA-N 0.000 description 1
- POJOORKDYOPQLS-UHFFFAOYSA-L barium(2+) 5-chloro-2-[(2-hydroxynaphthalen-1-yl)diazenyl]-4-methylbenzenesulfonate Chemical compound [Ba+2].C1=C(Cl)C(C)=CC(N=NC=2C3=CC=CC=C3C=CC=2O)=C1S([O-])(=O)=O.C1=C(Cl)C(C)=CC(N=NC=2C3=CC=CC=C3C=CC=2O)=C1S([O-])(=O)=O POJOORKDYOPQLS-UHFFFAOYSA-L 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- OTJZCIYGRUNXTP-UHFFFAOYSA-N but-3-yn-1-ol Chemical compound OCCC#C OTJZCIYGRUNXTP-UHFFFAOYSA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- VYXSBFYARXAAKO-WTKGSRSZSA-N chembl402140 Chemical compound Cl.C1=2C=C(C)C(NCC)=CC=2OC2=C\C(=N/CC)C(C)=CC2=C1C1=CC=CC=C1C(=O)OCC VYXSBFYARXAAKO-WTKGSRSZSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 230000002153 concerted effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 229940124447 delivery agent Drugs 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000438 effect on necrosis Effects 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010017007 glucose-regulated proteins Proteins 0.000 description 1
- 125000003827 glycol group Chemical group 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 230000009097 homeostatic mechanism Effects 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 230000009191 jumping Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 208000013469 light sensitivity Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000009061 membrane transport Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- IKEOZQLIVHGQLJ-UHFFFAOYSA-M mitoTracker Red Chemical compound [Cl-].C1=CC(CCl)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 IKEOZQLIVHGQLJ-UHFFFAOYSA-M 0.000 description 1
- 230000025608 mitochondrion localization Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000000329 molecular dynamics simulation Methods 0.000 description 1
- 230000037023 motor activity Effects 0.000 description 1
- 230000004987 nonapoptotic effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000003094 perturbing effect Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000002600 positron emission tomography Methods 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- TVDSBUOJIPERQY-UHFFFAOYSA-N prop-2-yn-1-ol Chemical compound OCC#C TVDSBUOJIPERQY-UHFFFAOYSA-N 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 231100000161 signs of toxicity Toxicity 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 150000003460 sulfonic acids Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000002691 unilamellar liposome Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
- C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
- C07D335/10—Dibenzothiopyrans; Hydrogenated dibenzothiopyrans
- C07D335/12—Thioxanthenes
-
- 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
- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
- C07F5/02—Boron compounds
- C07F5/022—Boron compounds without C-boron linkages
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
Definitions
- the present disclosure pertains to methods of opening a lipid bilayer by associating the lipid bilayer with a molecule that includes a moving component capable of moving (e.g., rotating) in response to an external stimulus; and exposing the molecule to an external stimulus.
- the exposing can occur before, during or after associating the molecule with the lipid bilayer.
- the exposing causes the moving component of the molecule to move. Thereafter, the movement facilitates the opening of the lipid bilayer. Additional embodiments of the present disclosure pertain to the aforementioned molecules.
- the molecules of the present disclosure may include various additional components.
- the molecules of the present disclosure may also include a base component that is capable of embedding with a lipid bilayer.
- Additional components of the molecules of the present disclosure can also include, without limitation, targeting agents for directing the molecule to a desired lipid bilayer, tracing agents for tracking the molecule, solubilizing agents for maintaining the water solubility of the molecule, active agents that may be releasable from the molecule, and combinations thereof.
- the lipid bilayer that is opened by the methods and molecules of the present disclosure is a component of cell membranes in vitro. In some embodiments, the lipid bilayer is a component of cell membranes in vivo.
- the molecules of the present disclosure are associated with lipid bilayers in vivo in a subject.
- the association step includes administering the molecule to the subject.
- the administered molecules may be utilized to treat a disease in a subject, such as skin-related cancers (e.g., skin cancers, colorectal cancers, oral cancers, and vaginal cancers).
- the lipid bilayers of the present disclosure may be exposed to various external stimuli.
- the external stimuli include an energy source, such as ultraviolet light, visible light, near-infra red light, a radio frequency energy source, a magnetic field, a two-photon energy source, an electric field, an electromagnetic field, and combinations thereof.
- the energy source includes ultraviolet light.
- the movement of the moving component of the molecules of the present disclosure facilitates the opening of the lipid bilayer by forming pores in the lipid bilayer.
- the opening of the lipid bilayer allows for the passage of materials through the lipid bilayer.
- the materials include, without limitation, analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof.
- the opening of the lipid bilayer allows for the passage of materials through the lipid bilayer and into cells for various purposes.
- FIG. 1 provides a scheme of a method of opening a lipid bilayer.
- FIGS. 2 A- 2 D- 2 provide illustrations of molecular motors and control molecules for disruption of lipid bilayers through molecular mechanical action.
- FIG. 2 A provides a schematic of molecular machines atop a cell membrane that open the membrane by UV-activated nanomechanical action.
- FIG. 2 B shows a representative molecular machine with a rotor portion that is light-activated to rotate relative to the larger bottom stator portion, where the addends (R) can be varied to provide the requisite solubility, fluorophores for tracking, or recognition sites for cellular targeting.
- R addends
- FIGS. 1 and 2 C shows representative structures of various nanomachines, including nanomachines 1 and 2 that bear fluorophores as pendants on the stator portions for tracking their movement, nanomachines 3 and 4 that have smaller molecular sizes with no stator-addended fluorophores for their tracking, control compound 5 which has a stator segment but no rotor and no capability of being UV-activated, and control compound 6 with a slow rotor.
- FIGS. 1 and 2 that bear fluorophores as pendants on the stator portions for tracking their movement
- nanomachines 3 and 4 that have smaller molecular sizes with no stator-addended fluorophores for their tracking
- control compound 5 which has a stator segment but no rotor and no capability of being UV-activated
- control compound 6 with a slow rotor.
- 2 D- 1 and 2 D- 2 show representative structures of additional nanomachines, including nanomachines 7 and 8 that are functionalized with the DGEA peptide sequence to target ⁇ 2 ⁇ 1 -integrin overexpressed in PC-3 human prostate cancer cells, and nanomachines 9 and 10 that are functionalized with the SNTRVAP peptide sequence to bind to the 78 kDa glucose regulated protein (GRP78) targeting castrate-resistant osteogenic prostate cancer receptors on PC-3 human prostate cancer cells.
- GRP78 glucose regulated protein
- Rotors in nanomachines 1-4 and 7-10 rotate at 2-3 MHz when activated with 355 to 365 nm light.
- Rotor in nanomachine 6 rotates at 1.8 revolutions per hour when activated with 355 to 365 nm light at 60° C., but only cis-trans isomerizes about the rotor-stator double bond at room temperature.
- FIG. 3 provides a schematic of dye releasing experiments upon UV illumination.
- FIGS. 4 A-K show data related to UV light-activated molecular motor 1 (illustrated in FIG. 2 C ) and its releasing of encapsulated dye molecules from synthetic bilipid vesicles through nanomechanical action.
- FIGS. 4 A-F show data related to BODIPY dye release from the vesicles.
- FIG. 4 A shows a dark-field image of the vesicles.
- FIG. 4 B shows a fluorescence image of molecular motor 1.
- FIG. 4 C shows fluorescence images of BODIPY dyes.
- FIG. 4 D shows co-localization of the images in FIGS. 4 A-C .
- FIG. 4 E shows the fluorescence image of the BODIPY dye as a function of UV-exposure time.
- FIGS. 4 G-J show control experiments with compound 16 (illustrated in FIG. 4 K ) instead of molecular motor 1.
- FIG. 4 G shows a dark-field image of the vesicles.
- FIG. 4 H shows a fluorescence image of compound 16. The image shows that 16 is incorporated into the lipid bilayer.
- FIG. 4 I shows a fluorescence image of the BODIPY dye and compound 16 as a function of UV-exposure time.
- FIG. 4 J shows normalized fluorescence intensity vs UV-exposure time of 20 vesicles from 5 different sets of movies.
- the scale bar in FIG. 4 A is 10 ⁇ m and is the same for FIGS. 4 B-D .
- FIG. 4 G and FIG. 4 H The scale bar in FIG. 4 I is 2 ⁇ m and is the same for all the figures in FIG. 4 E and FIG. 4 I .
- FIGS. 5 A-B show the optical properties of molecular machines 1 and 2.
- FIG. 5 A shows the UV/vis absorption and fluorescence spectra of molecular machine 1. Excitation was observed at 630 nm.
- FIG. 5 B shows the UV/vis absorption and fluorescence spectra of compound 2. Excitation was observed at 474 nm.
- FIGS. 6 A-D show images of NIH 3T3 cells in the presence of the fluorescent molecular machines 1 and 2, which were studied with UV activation to cause nanomechanical-induced entry of molecular machines 1 and 2 into the cells.
- FIG. 6 A shows images of cells exposed to nanomachine 2, including a left image (green, C loading 500 nM/2 h, ⁇ ex 514 nm, ⁇ em 520-540 nm, 2 mW); a middle image MitoTrackerRed (red, C loading 100 nM/30 min, ⁇ ex 543 nm, ⁇ em 550-600 nm, 0.5 mW); and a right image that represents the two merged transmission images verifying mitochondrial localization.
- FIG. 6 A shows images of cells exposed to nanomachine 2, including a left image (green, C loading 500 nM/2 h, ⁇ ex 514 nm, ⁇ em 520-540 nm, 2 mW); a middle image Mit
- FIG. 6 B shows images of cells exposed to nanomachine 1, including a left image (red, C loading 500 nM/1 h, ⁇ ex 633 nm, ⁇ em 650-700 nm, 1 mW); a middle image LysoTrackerGreen (green, C loading 200 nM/5 min, ⁇ ex 488 nm, ⁇ em 500-530 nm, 0.2 mW); and a right image that represents the two merged transmission images highlighting pit-like surface localization.
- FIG. 6 C shows a merged transmission (488 nm, 0.2 mW) images demonstration time dependent 1 internalization.
- UV-activation has been achieved using parallel ⁇ ex 355 nm, 20 mW 400 nJ/voxel total dwell time for the corresponding times noted in the images.
- FIG. 6 D shows fluorescent images demonstrating time-dependent dispersion of formed intracellular aggregates of 1 after a 1 hour incubation and wash cycles followed by UV-activation for the corresponding times noted in the images. All scale bars are 20 ⁇ m.
- FIGS. 7 A-E show the effects of nanomachines 3 and 4, and control molecule 5 on PC-3 cells upon UV-activation. The rate of necrotic cell death and permeabilization of analytes into the cells was recorded. The UV-exposure times are shown in each image.
- FIG. 7 A shows blank cells without molecular motors.
- FIG. 7 B shows cells exposed to nanomachine 3.
- FIG. 7 C shows cells exposed to nanomachine 4.
- FIG. 7 D shows cells exposed to control molecule 5. All the exposures occurred at 500 nM with 5 minute incubation before imaging.
- FIG. 7 A shows blank cells without molecular motors.
- FIG. 7 B shows cells exposed to nanomachine 3.
- FIG. 7 C shows cells exposed to nanomachine 4.
- FIG. 7 D shows cells exposed to control molecule 5. All the exposures occurred at 500 nM with 5 minute incubation before imaging.
- FIG. 7 E shows an identical imaging sequence using nanomachine 3 with the introduction of 100 nM PI (red, ⁇ ex 543 nm, ⁇ em 610-630 nm, 0.2 mW) confirming molecular mechanical cell permeabilization with intercalation of RNA and DNA primarily in the cell nuclei. All scale bars are 20 ⁇ m. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- FIGS. 8 A-D show interactions of compounds 3, 4 and 5 with NIH 3T3 cells, which lead to monitored necrosis. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm, ⁇ em 460-550 nm, 20 mW 400 nJ/voxel total dwell time, 1024 ⁇ 1024 pixel) images of NIH 3T3 cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes at 500 nM at 5 minute incubation time. The UV-exposure times are shown in each image.
- FIG. 8 A shows blank cells without molecular motors.
- FIG. 8 B shows cells with compound 3.
- FIG. 8 C shows cells with compound 4.
- FIG. 8 D shows cells with compound 5. All scale bars are at 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- the determination of onset (orange) and final stage (red) of necrosis are shown combining: 4 to 6 individual microscope slides with 5 to 6 FOV on each with an average 2.1 to 2.7 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. Table 2 provides more details.
- FIGS. 9 A-E show interactions of the cis-trans isomerizing 6 with NIH 3T3 and PC-3 showing no enhanced necrosis, and non-directional rotating demethylated 3 (Table 2) showing slowed necrosis.
- FIG. 9 A shows PC-3 cells without compound 6.
- FIG. 9 B shows NIH 3T3 cells without compound 6.
- FIG. 9 C shows NIH 3T3 cells with compound 6.
- FIG. 9 D shows PC-3 cells with compound 6.
- FIG. 9 E shows PC-3 cells with a demethylated version of compound 3 (no methyl group at the allylic position, so no unidirectional rotation). All scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- the determination of onset (orange) and final stage (red) of necrosis are shown by combining 4 to 5 individual microscope slides with 21 to 25 FOV on each with an average 2.5 to 3.1 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 10 A-E show the effects of UV-activated compounds 7 and 8 on PC-3 cells while monitoring necrotic cell death.
- FIG. 10 A shows blank cells without molecular motors.
- FIG. 10 B shows cells with compound 7 before washing.
- FIG. 10 C shows cells with compound 8 before washing.
- FIG. 10 D shows cells with compound 7 after washing.
- FIG. 10 E shows cells with compound 8 after washing. Scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- the determination of onset (orange) and final stage (red) of necrosis are shown combining 4 to 5 individual microscope slides with 5 to 7 FOV on each with an average 2.2 to 3.1 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 11 A-F provide data indicating that molecular motors 7 and 8 show an unexpectedly strong association with NIH 3T3 cells during nanomechanical-induced necrosis.
- UV-activated nanomechanical-accelerated necrosis by compounds 7 and 8 occurred in the same timeframe as with the PC-3 cells, indicating no desired selectivity of PC-3 over the NIH 3T3 cells studied in this Figure. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm.
- FIG. 11 A shows cells with compound 3.
- FIG. 11 B shows blank cells without nanomachines.
- FIG. 11 C shows cells with compound 7 before washing.
- FIG. 11 D shows cells with compound 8 before washing.
- FIG. 11 E shows cells with compound 7 after washing.
- FIG. 11 F shows cells with compound 8 after washing. Scale bars are 20 ⁇ m. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- onset (orange) and final stage (red) of necrosis are shown by combining 4 to 6 individual microscope slides with 5 to 7 FOV on each with an average 2.2 to 2.6 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 12 A-E show the study of targeted nanomechanical action of compounds 9 and 10 upon PC-3 cell necrosis. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm, ⁇ em 460-550 nm, 20 mW 400 nJ/voxel total dwell time, 1024 ⁇ 1024 pixel) images of PC-3 human prostate cancer cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes. PI was added to all the cell media. The UV-exposure times are shown in each image.
- FIG. 12 A shows blank cells without molecular motors.
- FIG. 12 B shows cells exposed to compound 9 without washing.
- FIG. 12 C shows cells exposed to compound 9 followed by washing.
- FIG. 12 D shows cells exposed to compound 10 without washing.
- FIG. 12 E shows cells exposed to compound 10 followed by washing. All scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- the determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.1 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 13 A-E show the study of nanomechanical action of compounds 9 and 10 upon PC-3 (targeted) and NIH 3T3 cells (untargeted) showing that PC-3 cell necrosis occurs faster than NIH 3T3 cell necrosis. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm, ⁇ em 460-550 nm, 20 mW 400 nJ/voxel total dwell time, 1024 ⁇ 1024 pixel) images of cancer cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes. The UV-activation times are noted in each image and 100 nM PI was in the medium.
- FIG. 13 A shows PC-3 blank cells without motors after 24 hours.
- FIG. 13 B shows PC-3 cells exposed to compound 9 and no washing after 24 hours.
- FIG. 13 C shows PC-3 cells exposed to compound 9, followed by washing, after 24 hours of incubation.
- FIG. 13 D shows PC-3 cells exposed to compound 10 without washing after 24 hours.
- FIG. 13 E shows NIH 3T3 cells exposed to compound 9 without washing after 24 hours. Scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.1 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 14 A-E show the study of the nanomechanical action of compounds 9 and 10 upon NIH 3T3 cells (untargeted) showing little enhanced rate of necrosis. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm, ⁇ em 460-550 nm, 20 mW 400 nJ/voxel total dwell time, 1024 ⁇ 1024 pixel) images of NIH 3T3 cells depicting time dependent UV-activated nanomechanical-induced cell morphological changes. The UV-activation times are noted in each image and 100 nM PI was in the medium.
- FIG. 14 A shows blank cells without motors.
- FIG. 14 A shows blank cells without motors.
- FIG. 14 B shows cells exposed to compound 9 by a 1 hour incubation and no washing.
- FIG. 14 C shows cells exposed to compound 9 by a 1 hour incubation followed by washing.
- FIG. 14 D shows cells exposed to compound 10 by a 1 hour incubation without washing.
- FIG. 14 E shows cells exposed to compound 10 by a 1 hour incubation followed by washing.
- Scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 4 to 6 FOV on each with an average 2.4 to 3.3 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 15 A-E show the study of nanomechanical action of compounds 9 and 10 upon CHO cells (untargeted) showing little enhanced necrosis. Shown are recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, ⁇ ex 355 nm. ⁇ em 460-550 nm, 20 mW 400 nJ/voxel total dwell time, 1024 ⁇ 1024 pixel) images of the study of compounds 9 and 10 in CHO cells depicting time dependent UV-activated nanomechanical-induced cell morphological changes. The UV-activation times are noted in each image and 100 nM PI was in the medium.
- FIG. 15 A shows blank cells.
- FIG. 15 B shows cells exposed to compound 9 without washing.
- FIG. 15 C shows cells exposed to compound 10 without washing.
- FIG. 15 D shows cells exposed to compound 9 by 30 minutes of incubation with cells after washing.
- FIG. 15 E shows cells exposed to compound 9 with 24 hours of incubation followed by washing of cells. Scale bars are 20 ⁇ m.
- the statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row.
- the determination of onset (orange) and final stage (red) of necrosis are shown by combining 3 to 5 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.2 cells per FOV.
- the displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details.
- FIGS. 16 A-B show whole-cell patch clamp studies of the dynamic effects of UV-induced molecular mechanical action of compound 3 upon HEK293 cells. Also shown are control studies using compound 3 without UV activation. UV-exposed rotor-free control molecule 5, and no molecular additives x. Compounds 3 and 5 were used at 1.0 ⁇ M concentrations.
- FIG. 16 A shows transmembrane currents in HEK293 cells showing that cells treated with UV (355 nm)-activated molecular motors 3 have inward currents consistent with membrane degradation (bottom trace). Without UV illumination, cells treated with compounds 3 or 5 show no change in membrane currents over the 4 minute recording period (top two traces).
- the white arrows highlight membrane blebbing that appears only in the cells treated with UV-activated compound 3.
- the scale bar represents 10 ⁇ m and is applicable for each micrograph.
- FIGS. 17 A-D show the consecutive monitoring of the interconversion of cis and trans isomers of compound 8.
- FIG. 17 A is a chromatogram of one of the isomers immediately after separation.
- FIG. 17 B is a chromatogram of the fraction after being stored in the freezer for two weeks.
- FIG. 17 C is a chromatogram of the fraction after being exposed to room light for 20 minutes.
- FIG. 17 D is a chromatogram of the fraction after being irradiated with laboratory TLC UV light for 15 minutes.
- FIGS. 18 A-C show data related to monitoring the UV-activated nanomechanical action of compound 3 upon exposure to NIH 3T3 cells using a conventional CW mercury-arc excitation source equipped with an epi-fluorescence setup consisting of a Zeiss Axiovert 200M inverted microscope. Shown are live cell microscopy images of a selected NIH 3T3 cell pre-incubated with 500 nm of compound 3 for 30 minutes in live cell imaging cell culture media (Method A), including; (1) (green) UV-induced mitochondrial autofluorescence ( ⁇ ex BP365/50 nm.
- FIGS. 19 A-D provide a demonstration that a two-photon illumination in the near-infra red (IR) region can activate molecular motors 3 or 8 and result in PI dye entering NIH 3T3 or PC-3 cells.
- FIG. 19 A shows images of cells in live cell media with no molecular motors present.
- FIG. 19 B shows images of identical cells but with a 1 ⁇ M control molecular motor 5 using Method A.
- FIG. 19 C shows images of identical cells but with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 19 D shows images of PC-3 cells with 1.0 ⁇ M of molecular motor 8 present using Method B.
- the scale bars correspond to 20 ⁇ m.
- FIGS. 20 A-L provide an additional demonstration that a two-photon illumination in the near-infra red (IR) region can activate the molecular motors 3, 8 or 9 and result in PI dye entering NIH 3T3 or PC-3 cells.
- FIG. 20 A shows images of control NIH 3T3 cells in live cell media under two-photon illumination with no molecular motors present.
- FIG. 20 B shows images of identical NIH 3T3 cells with 1 ⁇ M of control 5 present using Method A.
- FIG. 20 C shows images of identical NIH 3T3 cells with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 20 D shows images of NIH 3T3 cells with 1 ⁇ M of molecular motor 8 present using Method B.
- FIG. 20 E shows images of identical control PC 3 cells under two-photon illumination with no molecular motors present.
- FIG. 20 F shows images of identical PC 3 cells with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 20 G shows images of identical PC-3 cells with 1 ⁇ M of control 5 present using Method A.
- FIG. 20 H shows images of identical PC-3 cells with 1 ⁇ M of molecular motor 3 present using Method B.
- FIG. 20 I shows images of identical PC-3 cells with 1 ⁇ M of molecular motor 8 present using Method A.
- FIG. 20 J shows images of identical PC-3 cells with 1 ⁇ M of molecular motor 9 present using Method A.
- FIG. 20 K shows images of identical PC3 cells with 1 ⁇ M of molecular motor 8 present using Method B.
- FIG. 20 L shows images of identical PC-3 cells with 1 ⁇ M of molecular motor 9 present using Method B.
- the scale bars correspond to 20 ⁇ m.
- the molecular photoswitch azobenzene has been adapted and used as a photochromic K + channel opener, as an optical controller of insulin secretion, and for restoration of light sensitivity in blind retinae.
- the present disclosure pertains to methods of opening a lipid bilayer.
- the methods of the present disclosure include steps of associating the lipid bilayer with a molecule that includes a moving component capable of moving in response to an external stimulus (step 10), and exposing the molecule to an external stimulus (step 12).
- the exposure of the molecule to the external stimulus causes the moving component of the molecule to move (step 14).
- the movement of the moving component of the molecule facilitates the opening of the lipid bilayer (step 16).
- the opening of the lipid bilayer can be used to facilitate the passage of various materials through the lipid bilayer (step 18). Additional embodiments of the present disclosure pertain to the aforementioned molecules for opening a lipid bilayer.
- FIG. 2 A Examples of the methods and molecules of the present disclosure are also depicted in FIG. 2 A .
- molecule 22 with moving component 24 becomes associated with lipid bilayer 20.
- the molecule is exposed to light irradiation (i.e., an external stimulus).
- the light irradiation causes moving component 24 of molecule 22 to move (i.e., rotate in this embodiment) and facilitate the opening of lipid bilayer 20 through the formation of pores 26.
- the present disclosure can have numerous embodiments.
- various molecules may become associated with various types of lipid bilayers in various manners.
- the molecules may be exposed to various external stimuli in order to open the lipid bilayers through various mechanisms.
- the molecules and methods of the present disclosure can be utilized to pass various materials through the opened lipid bilayers for various purposes and applications.
- the methods of the present disclosure can utilize various types of molecules for opening lipid bilayers. Additional embodiments of the present disclosure pertain to the aforementioned molecules for opening lipid bilayers.
- the molecules of the present disclosure generally include a moving component capable of moving in response to an external stimulus.
- the moving components of the present disclosure can include one or more conjugated systems.
- the wavelength of the conjugated system may shift to the visible region, thereby making the moving component activatable to visible light.
- the molecules of the present disclosure also include a base component that is capable of embedding with a lipid bilayer.
- the moving component is also capable of embedding with the lipid bilayer.
- the base component is also capable of moving in response to an external stimulus.
- the molecules of the present disclosure also include one or more targeting agents.
- the targeting agent is capable of directing the molecule to a specific type of a lipid bilayer, such as a lipid bilayer associated with cell membranes of particular cells, organs, or tissues.
- the targeting agent is capable of binding to a receptor on a lipid bilayer of a cell membrane.
- the molecules of the present disclosure may be associated with various types of targeting agents.
- the targeting agent includes, without limitation, amino acids, peptides, proteins, aptamers, antibodies, small molecules, carbohydrates, polysaccharides, and combinations thereof.
- the targeting agent includes peptides.
- the targeting agent includes antibodies, such as monoclonal antibodies.
- the targeting agents may be used to target specific cell types, such as cancer cells (e.g., cancer cells associated with a specific type of cancer, such as skin-related cancers), fat (adipocyte) cells, or diseased cells.
- cancer cells e.g., cancer cells associated with a specific type of cancer, such as skin-related cancers
- fat (adipocyte) cells e.g., hematomas, or other cells.
- the targeted cells have an overexpressed and specific cell surface receptor that is recognized by the targeting agents.
- the molecules of the present disclosure may also include one or more tracing agents.
- the tracing agent can be utilized to track the association of molecules with a lipid bilayer.
- the molecules of the present disclosure may be associated with various types of tracing agents.
- the tracing agents may be detectable by magnetic resonance imaging (MRI), positron emission tomography (PET), or other imaging techniques.
- the tracing agents include, without limitation, fluorophores, chromophores, dyes, radio-labeled molecules, radioactive nuclei, high contrast agents, gadolinium, gallium, thallium, fluorinated compounds, and combinations thereof.
- the molecules of the present disclosure also include one or more solubilizing agents.
- the solubilizing agents help maintain the water solubility of the molecule.
- the molecules of the present disclosure may be associated with various types of solubilizing agents.
- the solubilizing agents include, without limitation, peptides, glycols, alcohols, carboxylates, polysaccharides, salts, acids, polyethers, polyethylene glycols (PEGs), carbohydrates, and combinations thereof.
- the solubilizing agents include glycols, such as polyethylene glycol units.
- the solubilizing agents include alcohols, such as polyvinyl alcohol and polyols.
- the solubilizing agents include carboxylates, such as carboxylate moieties.
- the solubilizing agents include acids, such as sulfonic acids.
- the solubilizing agents include salts, such as ammonium salts.
- the molecules of the present disclosure may also include one or more active agents.
- the molecules of the present disclosure may be associated with one or more active agents in a releasable manner.
- the molecules of the present disclosure are releasably associated with one or more active agents through a cleavable bond, such as an ester linkage (e.g., cleavable by an esterase), an amide linkage (e.g., cleavable by an amidase), or a photolabile linkage (e.g., cleavable by UV light).
- the molecules of the present disclosure are releasably associated with one or more active agents such that the one or more active agents are released from the molecules once the molecules facilitate the opening of the lipid bilayer or enter cells.
- the molecules of the present disclosure may be associated with various types of active agents.
- the active agents include, without limitation, drugs, peptides, polypeptides, nucleotides. DNA, RNA, siRNA, enzymes, and combinations thereof.
- the active agents include drugs, such as anti-cancer drugs.
- the active agents include peptides. The use of additional active agents can also be envisioned.
- the molecules of the present disclosure include the following structure (depicted as structure 1):
- Region A in structure 1 includes moving component R 3 , which is capable of moving in response to an external stimulus.
- Region B in structure 1 includes a base component.
- the base component is capable of embedding with a lipid bilayer.
- the moving component is capable of embedding with a lipid bilayer.
- the moving and base components can embed with a lipid bilayer.
- Structure 1 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents. In some embodiments, the other components can also embed with the lipid bilayer.
- R 1 and R 2 in structure 1 can include various groups and moieties.
- R 1 and R 2 can each independently include, without limitation, hydrogen, alkanes, alkenes, alkynes, carboxyl groups, ketone groups, alkoxy groups, methoxy groups, ethers, nitro groups, nitriles, sulfates, sulfonates, halogens, amine groups, amide groups, alcohols, aromatic groups, aryl groups, phenyl groups, annulated rings, carbohydrates, polysaccharides, peptides, targeting agents, tracing agents, fluorophores, solubilizing agents, active agents, and combinations thereof.
- R 1 and R 2 can each include annulated rings.
- the annulated rings when aromatic or further pi-electron-conjugated, can facilitate the activation of the molecules of the present disclosure by a lower energy source, such as visible light.
- X in structure 1 can also include various groups and moieties.
- X can include, without limitation, S, CH 2 , O, and combinations thereof. In some embodiments, X includes S.
- R 3 in structure 1 can also include various structures.
- R 3 includes the following structure (depicted as structure 2):
- R 3 in structure 1 includes the following structure (depicted as structure 3):
- the moving components or molecules may have one or more annulated rings, such has annulated aromatic rings. Due to the additional annulated aromatic rings, the light absorbance and emission spectra of a molecule's moving component may undergo bathochromic shifts in some embodiments, thereby being excited in the visible region at 400 nm or higher wavelengths (e.g., at least 500 nm or even 700 nm).
- the presence of additional annulated aromatic rings on a molecule or a moving component may facilitate the excitation of the moving component in the near infrared (IR) region at greater than 700 nm.
- IR near infrared
- the energy that the moving component exerts on the lipid bilayer decreases. Therefore, in some embodiments, an assessment of the energy requirements for lipid bilayer disruption followed by correlation to the excitation and emission energies of the molecule may be required. The Examples provide various methods for such energy calculation.
- the molecules of the present disclosure include the following structure (depicted as structure 4):
- Region A in structure 4 includes a moving component capable of moving in response to an external stimulus.
- Region B in structure 4 includes a base component that is capable of embedding with a lipid bilayer.
- Structure 4 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents.
- R 1 and R 2 can include various moieties and functional groups that were described previously.
- the molecules of the present disclosure include the following structure (depicted as structure 5):
- Region A in structure 5 includes a moving component capable of moving in response to an external stimulus.
- Region B in structure 5 includes a base component that is capable of embedding with a lipid bilayer.
- Structure 5 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents.
- R 1 and R 2 can include various moieties and functional groups that were described previously.
- FIGS. 2 C- 2 D -2 e.g., molecules 1-4 and 6-10.
- aforementioned structures 1-5 are different from molecules 1-5 depicted in FIG. 2 C .
- the molecules and methods of the present disclosure can be utilized to open various types of lipid bilayers.
- the lipid bilayers may be components of cell membranes, such as external or internal cellular membranes.
- the lipid bilayers may be components of an organelle, such as the mitochondria.
- the lipid bilayers may be components of a nuclear membrane.
- the lipid bilayers may be components of cell membranes in vivo, such as cell membranes of a tissue or organ in a subject (e.g., a human being).
- the cell membranes may be components of various cell types of interest, such as cancer cells, tumor cells, diseased cells, fat cells, and combinations thereof.
- the lipid bilayers may be components of cell membranes in vitro, such as cell membranes in a cell culture medium. In some embodiments, the lipid bilayers may be components of vesicles, such as synthetic vesicles in vitro. The use of additional lipid bilayers can also be envisioned.
- the molecules of the present disclosure may become associated with lipid bilayers in various manners. For instance, in some embodiments, the molecules of the present disclosure become embedded within the lipid bilayer. In some embodiments, the molecules of the present disclosure are inserted into the lipid bilayer. In some embodiments, the molecules of the present disclosure are placed on surfaces of the lipid bilayer.
- the molecules of the present disclosure may become associated with lipid bilayers by various steps. For instance, in some embodiments, the molecules of the present disclosure become associated with lipid bilayers by exposing the lipid bilayers to the molecules. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers by incubating the lipid bilayers with the molecules. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers by contacting the lipid bilayers with the molecules.
- the molecules of the present disclosure become associated with lipid bilayers in vitro. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers in vivo in a subject (e.g., a human being). In some embodiments, the molecules of the present disclosure become associated with lipid bilayers in vivo in a subject by administering the molecules to the subject.
- the administration occurs by a method that includes, without limitation, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, intrathecal injection, topical administration, central administration, peripheral administration, and combinations thereof.
- the molecules of the present disclosure may be co-administered to a subject with additional materials, such as active agents.
- the administered molecules of the present disclosure may be releasably linked to an active agent.
- the administered molecules may be utilized to treat a disease in a subject, such as skin-related cancers.
- the skin-related cancers include, without limitation, skin cancer (e.g., melanoma), colorectal cancers, oral cancers, vaginal cancers, and combinations thereof.
- the molecules of the present disclosure may be administered to a subject through intravenous administration (e.g., intravenous injection).
- intravenous administration e.g., intravenous injection
- the molecules of the present disclosure may be administered onto a skin of subject through subcutaneous (i.e., subdermal) administration (e.g., subcutaneous injection).
- subcutaneous administration e.g., subcutaneous injection
- the subdermal administration can occur in the presence of a skin-penetrating material, such as dimethylsulfoxide, thereby carrying the molecule through the skin.
- the molecule e.g., the molecule and its associated or co-administered active agent
- the dermal transport agents can also facilitate transport of the molecules of the present disclosure (e.g., the molecule and its associated or co-administered active agents) through the vaginal, colorectal, oral and gastrointestinal layers to facilitate transport of the molecules to their sites of interest before or during activation by exposure to an external stimulus (e.g., light or other external stimuli).
- an external stimulus e.g., light or other external stimuli
- the molecules of the present disclosure may be co-administered to a subject along with an energy source capable of providing an external stimulus.
- the energy source includes a light source, such as an LED lamp in capsule form.
- the molecules of the present disclosure may be co-administered to a subject along with an energy source (e.g., LED lamp) and an active agent (e.g., peptide-based drug).
- a capsule that contains an LED lamp and the molecules of the present disclosure (i.e., nanomachines) and an active agent (e.g., a drug associated with the molecule or co-administered with the molecule, such as a peptide-based drug) can be dissolved in the small intestines such that the LED lamp activates the molecules (i.e., nanomachines) which aid in opening the gastrointestinal lining to permit the active agent to enter the bloodstream.
- the aforementioned administration can occur in conjunction with an epithelial transport agent. Additional administration methods can also be envisioned.
- the lipid bilayers of the present disclosure may be associated with the molecules of the present disclosure for various periods of time. For instance, in some embodiments, the association takes place from about 1 minute to about 48 hours. In some embodiments, the association takes place from about 5 minutes to about 48 hours. In some embodiments, the association takes place from about 5 minutes to about 2 hours.
- the lipid bilayers of the present disclosure may become associated with various concentrations of the molecules of the present disclosure.
- the molecule concentrations range from about 10 nM to about 10 ⁇ M.
- the molecule concentrations range from about 100 nM to about 1 ⁇ M.
- the molecule concentrations range from about 100 nM to about 500 nM.
- the molecule concentrations range from about 100 nM to about 200 nM.
- the molecule concentrations are at least about 100 nM.
- the molecules of the present disclosure may be exposed to various external stimuli in order to cause the movement of their moving component.
- the molecules of the present disclosure are exposed to an external stimulus that includes an energy source.
- the energy source includes, without limitation, ultraviolet (UV) light, visible light, near-infra red (IR) light, a radio frequency (RF) energy source, a two-photon energy source, an electric field, a magnetic field, an electromagnetic field, and combinations thereof.
- the energy source includes ultraviolet light.
- the energy source is in the form of electromagnetic radiation.
- a two-photon energy source is utilized to provide a very focused area of exposure.
- the energy source includes two photons of near-infra red light.
- the near-infra red light activates the molecules of the present disclosure by the use of two photons at about 710 nm.
- the molecules of the present disclosure are exposed to an energy source at various wavelengths.
- the wavelength of the energy source ranges from about 355 nm to about 365 nm.
- the wavelength of the energy source ranges from about 500 nm to about 610 nm.
- the wavelength of the energy source ranges from about 600 nm to about 750 nm. Additional wavelength ranges can also be envisioned.
- the molecules of the present disclosure may be exposed to an external stimulus for various periods of time.
- the exposure time may be from about 1 second to about 600 seconds.
- the exposure time may be from about 1 second to about 400 seconds.
- the exposure time may be from about 1 second to about 300 seconds.
- the exposure time may be from about 1 second to about 200 seconds.
- the exposure time may be from about 1 second to about 60 seconds.
- the exposure time may be from about 1 second to about 30 seconds. Additional exposure times can also be envisioned.
- the molecules of the present disclosure may be exposed to an external stimulus at various periods of time. For instance, in some embodiments, the exposing occurs after the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs before the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs while the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs before or during the association of the molecule with the lipid bilayer.
- the molecules of the present disclosure may be exposed to external stimuli in various environments.
- the molecules of the present disclosure are exposed to an external stimulus in vitro.
- the in vitro environment may be a cell culture medium that contains lipid bilayers as components of cell membranes.
- the molecules of the present disclosure are exposed to an external stimulus in vivo.
- the in vivo environment may be the organ or tissue of a subject that has been administered with the molecules of the present disclosure.
- the in vivo environment may be the skin of a subject that has been administered with the molecules of the present disclosure.
- the exposure of the molecules of the present disclosure to an external stimulus can have various effects on the molecules.
- the exposure causes the moving component of the molecule to move in various manners in response to the external stimulus.
- the movement includes, without limitation, rotation, flapping, jumping, and combinations thereof.
- the movement includes flapping.
- the movement is confined to the moving component of the molecule. In some embodiments, the movement occurs throughout the entire molecule.
- the movement includes rotation.
- the moving components of the molecules of the present disclosure can rotate in various manners. For instance, in some embodiments, the moving component of the molecules of the present disclosure rotates in a unidirectional manner. In some embodiments, the moving component rotates in a non-reciprocating unidirectional manner. In some embodiments, the moving component rotates in a bi-direction manner. In some embodiments, the moving component rotates relative to a base component of the molecule.
- the moving component of the molecules of the present disclosure can rotate for various degrees in response to an external stimulus. For instance, in some embodiments, the moving component rotates from about 45 degrees to about 360 degrees. In some embodiments, the moving component rotates from about 60 degrees to about 180 degrees. In some embodiments, the moving component rotates for at least about 180 degrees. In some embodiments, the moving component rotates for at least about 360 degrees.
- the moving component of the molecules of the present disclosure can also rotate at various rates in response to an external stimulus. For instance, in some embodiments, the moving component of the molecules of the present disclosure can rotate at rotation rates of about 1-10 MHz. In some embodiments, the moving component of the molecules of the present disclosure can rotate at rotation rates of about 2-3 MHz.
- the moving component of the molecules of the present disclosure can rotate at 1-10 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at 1-5 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at 1-2 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at about 1.8 revolutions per hour.
- the movement of the moving component of the molecules of the present disclosure can have various effects on lipid bilayers.
- the movement facilitates the opening of the lipid bilayers in various manners.
- the movement of the moving component of the molecules of the present disclosure changes the conformation of the molecule (e.g., change in the conformation of molecule 22, as depicted in FIG. 2 A ). Thereafter, the change in the conformation of the molecule leads to the opening of the lipid bilayer. In some embodiments, the movement produces a tangential mechanical force that leads to the opening of the lipid bilayer.
- the movement of the moving component of the molecules of the present disclosure facilitates the opening of lipid bilayers by disrupting the lipid bilayers. In some embodiments, the movement facilitates the opening of the lipid bilayers by dislocation of lipid bilayer molecules. In some embodiments, the movement facilitates the opening of the lipid bilayers by causing rupture or degradation of the lipid bilayers.
- the opening of lipid bilayers by the molecules of the present disclosure may result in the formation of various structures.
- the lipid bilayers are opened by forming pores (e.g., pores 26, as depicted in FIG. 2 A ).
- the formed pores may be transient.
- the formed pores may be permanent.
- the formed pores may have various diameters. In some embodiments, the pore diameters range from about 10 nm to about 500 ⁇ m.
- the methods and molecules of the present disclosure may be utilized to open various types of lipid bilayers for various applications.
- the methods and molecules of the present disclosure can be utilized to open lipid bilayers for passage of various materials through the lipid bilayer.
- materials can include, without limitation, analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof.
- the materials can include nucleotides, such as siRNA, DNA, RNA, and combinations thereof.
- the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into cells. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and out of cells. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into vesicles. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and out of vesicles.
- the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into organelles of cells, such as the mitochondria. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into the nucleus of cells.
- the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into cells in order to induce various effects on the cells.
- induced effects can include, without limitation, cell death, necrosis, disease treatment, and combinations thereof.
- effects can occur in vitro (e.g., in a cell culture medium) or in vivo (e.g., in a subject).
- the methods and molecules of the present disclosure can be utilized to open lipid bilayers of a cell membrane in a cell culture medium.
- Such methods can include associating the lipid bilayers of the cell membranes with the molecules of the present disclosure and exposing the molecules to an external stimulus in the presence of various materials (e.g., analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof) such that the materials enter the cells after exposure and induce various effects on the cells (e.g., cell death, necrosis, disease treatment, and combinations thereof).
- materials e.g., analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof
- the methods and molecules of the present disclosure can be utilized to open lipid bilayers of a cell membrane associated with an organ or a tissue in a subject by administering the molecules of the present disclosure to the subject such that the lipid bilayers of the cell membranes of the desired organ or tissue become associated with the molecules of the present disclosure.
- the desired organ or tissue may be exposed to an external stimulus in the presence of various co-administered materials (e.g., analytes, active agents, drugs, nucleotides.
- DNA DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof) such that the materials enter the cells of the tissue or organ after exposure and induce various effects (e.g., cell death, necrosis, disease treatment, and combinations thereof).
- the methods and molecules of the present disclosure can be utilized for the sculpting of cells, such as fat (adipocyte) cells.
- the molecules of the present disclosure may be administered (e.g., subcutaneously injected) under the skin of a subject where fat cells reside (or administered through other methods, such as transdermal administration or intravenous injection).
- An external stimulus e.g., light or other external stimuli, such as radiofrequency fields, electric fields or magnetic fields
- irradiation e.g., UV, visible (e.g., red light) or two-photon near-infra red irradiation
- the aforementioned steps may be repeated for additional sculpting.
- the sculpting of cells in accordance with the methods of the present disclosure can be utilized to selectively remove cells (e.g., fat cells) from a desired area.
- the methods and molecules of the present disclosure can be utilized to treat skin-related cancers, such as skin cancer (e.g., melanoma), colorectal cancers, oral cancers, and vaginal cancers.
- the molecules of the present disclosure are administered to the affected cells (e.g., via subcutaneous injection or oral administration).
- An energy source e.g., a light source, such as a light source via an endoscope or an administered pill
- an external stimulus e.g., UV irradiation
- a UV light source e.g., at wavelengths of about 350-360 nm
- a near-infra red light source could be utilized to irradiate the cells.
- the methods and molecules of the present disclosure can be utilized to facilitate the uptake of various active agents (e.g., peptide-based drugs) by a subject.
- the active agent may be co-administered to a subject along with the molecules of the present disclosure and an energy source that is capable of providing an external stimulus (e.g., an LED light).
- the molecules of the present disclosure may be associated with the active agents and co-administered with the energy source.
- the co-administration occurs through the use of a carrier, such as a capsule.
- the carrier may release the active agent (e.g., peptide-based drug), the molecule (i.e., the nanomachine), and the energy source (e.g., an LED light) near a particular cellular region, tissue or organ of the subject (e.g., the jejunum). Thereafter, the molecule associates with lipid bilayers and is activated by the energy source. This in turn facilitates the lipid bilayers of the particular cellular region, tissue or organ to open and uptake the active agents.
- the active agent e.g., peptide-based drug
- the molecule i.e., the nanomachine
- the energy source e.g., an LED light
- nanomechanical action can (a) induce the diffusion of analytes out of synthetic vesicles, (b) enhance diffusion of traceable molecular machines into and within live cells, (c) induce necrosis, (d) introduce analytes into live cells, and (e) be selectively targeted to specific live cell-surface recognition sites through nanomachines bearing short peptide addends.
- Applicants demonstrate that, beyond in vitro applications demonstrated in this Example, in vivo use can follow, especially through the use of two-photon-, near-infrared- and radio-frequency-activated domains.
- FIG. 2 A A scheme for nanomechanical action upon a lipid bilayer is shown in FIG. 2 A and the general design of a molecular machine suitable for transport though a lipid bilayer is shown in FIG. 2 B .
- these include molecular motors bearing fluorophores for tracking (1 and 2), smaller nanomachines (3 and 4), a control that bears a stator but no rotor (5), a control analogue (6) that can only undergo cis-trans isomerization (flapping) at room temperature, and targeting systems that bear peptide sequences for binding to specific cell-surface receptors (7-10).
- molecular machine 1 displays enhanced diffusion in solution when the fast light-driven motor is activated by 355-365 nm UV light.
- Method A the molecular motors were loaded into the cell media and the imaging was initiated within 5 minutes to 24 hours.
- Method B the molecular motors were loaded into the cell media, incubated for 30 minutes to 24 hours, and then washed three times with fresh molecular motor-free media before imaging.
- Nanomachine 2 enters the cell and localizes in the mitochondria ( FIG. 6 A ). Conversely, nanomachine 1, when introduced to cells, displays pit-like cell surface localization ( FIG. 6 B ) and later, at 4 hour, small ⁇ 1 ⁇ m aggregates are seen inside the cytoplasm.
- the NIH 3T3 cells in the presence of the nanomachines were then studied with concomitant UV activation.
- UV-induced motor activation for 150 seconds (355 nm) 1, introduced by Method A, was found to cross the cell membrane, and it was internalized into cells in a time-dependent manner, displaying fast accelerated intracellular motion, compared to natural homeostatic cellular organelle movement in the absence of UV-nanomechanical activation ( FIG. 6 C ).
- Combined controlled time and UV-exposure-dependent experiments indicate that the small aggregates of 1 inside the cytoplasm dissolve or burst with further increasing of fluorescence signal in the cytoplasm ( FIG. 6 D ).
- nanomachine trafficking can be facilitated and precisely observed.
- UV-induced (355 nm) PC-3 necrosis is not initiated until approximately scan 20 (corresponding to 300 second continuous scanning UV exposure, 400 nJ/voxel total dwell time), characterized by massive disruption and rupture of the mitochondrial network and subsequent well-pronounced auto-fluorescent signal detectable in the nucleoli and nucleus wall ( FIG. 7 A ). This is observable by the nucleoli membrane permeabilization and DNA damage at the onset of necrosis.
- the UV-induced necrosis reaches final stages at approximately scan 40 (corresponding to 600 seconds) that is consistent with characteristic extracellular membrane rupture and homogenous cytosolic auto-fluorescence, indicating loss of cellular organelle boundaries.
- b FOV field of view.
- c Each individual statistical analysis have been executed on N number of total cells per corresponding experiment (no data was ever excluded and all images were used without post-processing) according to a standard Gaussian (2-tailed) fit.
- d Reported are the averaged time of death.
- e The percentage is in acceleration. For example, 50% means cells die 50% faster when rotors are present with UV exposure verses UV upon blank cells, the latter meaning that no nanomachines are present. Zero percent means blank reference time or no observed acceleration in necrosis time.
- f Standard deviation is calculated for time of full necrosis where the minimum precision and experimental error, due to the nature of microscopic imaging sequence, is 15 s.
- Nanomachine 4 bearing the larger aryl sulfonate moieties, might have been inhibited from having its rotor interact well with the cell membranes, or the addends themselves sterically encumbered their rotation near the membrane.
- control molecule 5 was studied to ensure that the rotary action is preferred for the bilayer perturbations. Using Method A on both PC-3 and NIH 3T3 cells, which has the same homopropargylic alcohol stator moieties as does 3, control molecule 5 shows no effect on necrosis upon standardized UV-exposure ( FIGS. 7 D and FIGS. 8 A-D ). This further suggests that the accelerated cell death seen with 3 was not primarily due to the short exposure to UV light or subsequent thermal processes, similar to Applicants' earlier synthetic bilayer studies.
- Applicants further confirmed the nanomechanical opening and subsequent permeabilization of the membrane by adding a dye to the cell medium to assess its exogenous entry into the cells that might be afforded by nanomechanical action.
- a dye to the cell medium to assess its exogenous entry into the cells that might be afforded by nanomechanical action.
- PI propidium iodide
- PI is a fluorescent intercalating agent that is not internalized by healthy cells, and it is non-toxic as shown by Applicants' molecular machine-free controls.
- FIG. 7 E Upon membrane disruption by nanomechanical action of 3 ( FIG. 7 E ).
- PI enters the cell travels to RNA- and DNA-rich areas where it intercalates and its excitation maximum subsequently displays ⁇ 30 nm bathochromic shift (from 535 to 565 nm) accompanied by a parallel hypsochromic emission maximum shift (from 617 nm to 600 nm).
- RNA- and DNA-induced PI fluorescence is detected between 600 and 630 nm, allowing time-dependent light-activated molecular motor-induced cell permeabilization to be confirmed. Further, PI was used to follow membrane damage that is due to UV-activated nanomachine activity leading to necrosis. Since the entry of the PI is on a relatively short time scale compared to the time of cell division, the cell has insufficient time to adopt programmed cell death (apoptosis). This was confirmed using Annexin V, an apoptosis-specific stain where no relevant fluorescence from this dye was observed throughout the course of the experiments.
- the peptide-bearing structures (7-10) were investigated to target specific cells for nanomachine-activated necrosis.
- the targeted cell line was PC-3 while NIH 3T3 and CHO cells were used as non-targeted controls. No selectivity was observed with the shorter peptide targeting moieties 7 and 8 (Table 2 and FIGS.
- inward currents in HEK293 cells appeared between 40 and 60 seconds after exposure to UV illumination.
- the slow rise in inward current during illumination suggests an accumulation of many small pores or increasing pore sizes.
- the stress applied on the membrane would be 540 mN m ⁇ 1 , far exceeding the requisite rupture stress for most bilipid membranes of 1-30 mN m ⁇ 1 .
- nanomechanical action can generate a concerted motion upon a 1-nm-long molecular rotor that will severely dislocate the membrane molecules, while other light absorbing molecules will merely dissipate the absorbed energy in random motions of atoms in the molecule, underscoring the efficacy of the nanomechanical effect for membrane disruption.
- TLC Thin layer chromatography
- DGEA-alkyne peptide 14 was synthesized manually using standard solid-phase Fmoc protocols with Fmoc-amino acids and 4-pentynoic acid and was prepared as a C-terminal amide using Rink amide MBHA resin.
- Each acylation with Fmoc-amino acids or 4-pentynoic acid was performed using HATU (4 equiv) and N,N-diisopropylethylamine (DIPEA) (4 equiv) for 45 minutes in dimethylformamide (DMF) at room temperature, followed by Fmoc deprotection with 20% piperidine in DMF. DMF was used to wash the resin between each acylation and deprotection step.
- DIPEA N,N-diisopropylethylamine
- Sample preparation involved dissolving the reaction mixture into 1 mL of a 1:1 mixture of H 2 O and CH 3 CN, followed by vigorous shaking and sonication. The mixture was then centrifuged at 14.000 rpm for 10 minutes to obtain a fully transparent supernatant for HPLC injection. This process was repeated on the pellet to extract as much product as possible, until the obtained supernatant resulted in no peaks in the HPLC chromatogram.
- a gradient elution system containing 0.1% TFA in water and ACN, ramping from 20% to 90% of acetonitrile over 23 minutes with a flow rate of 2.5 mL/min at 40° C. was employed, which resulted in a baseline resolution of chromatogram peaks.
- Sample preparation involved dissolving the reaction mixture into 1 ml of 2:1 mixture of CH 3 CN and (CH 3 ) 2 SO, followed by vigorous shaking and sonication. The mixture was then centrifuged at 14,000 rpm for 10 minutes to obtain a fully transparent supernatant for HPLC injection. This process was repeated on the pellet to extract as much product as possible, until the obtained supernatant resulted in no peaks in the HPLC chromatogram.
- Premixed and dried synthetic phospholipid blend [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at a ratio of 5:3:2] were stored in a ⁇ 20° C. freezer before use.
- DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
- DOPS 1,2-dioleoyl-sn-glycero-3-phospho-L-serine
- DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
- FIGS. 4 A-D show that both BODIPY and compound 1 were localized in the vesicle.
- the vesicles were then immobilized on a piece of cover-glass for the UV activation and releasing experiments.
- the UV light was provided by a UV LED at 365 nm with an intensity of ⁇ 10 W ⁇ cm 2 and analysis was done from 0 to 10-15 minutes.
- the fluorescence intensity of the BODIPY dye molecules in single vesicles was monitored using a confocal fluorescence microscope by continuously scanning a small area (20 ⁇ 20 ⁇ m 2 ).
- FIG. 4 E shows the normalized fluorescence intensity drop of 20 vesicles from 6 different sets of movies. Since the absolute intensity of the vesicles varied from vesicle to vesicle. Applicants normalized all the traces according to their initial intensities and analyzed the percent intensity drop. The error bar stands for the standard deviation of the normalized intensities. The first data point does not have an error bar. The average fluorescence intensity drop of 20 vesicles was 70 ⁇ 6% (mean ⁇ standard deviation) in the first 15 minutes of UV exposure. This is an observation based on many motor and dye molecules on individual vesicles. Even though the “gate” opening process is fast, the loss of dye molecules and their corresponding fluorescence is gradual and continuous, spanning a period as long as ⁇ 15 minutes.
- Compound 16 has two conjugated BODIPY molecules embedded in its molecular structure. However, its excitation and emission are both red-shifted by ⁇ 70 nm as compared to those of isolated BODIPY molecules. Thus, its fluorescence is negligible to that from the isolated BODIPY molecules used as the probes when excited at 488 nm and collected at 535 t 25 nm.
- FIGS. 4 G-H show that compound 16 was localized in the vesicles.
- the fluorescence image of compound 16 was collected in the epi-fluorescence mode using 545 ⁇ 30 nm excitation and 605 ⁇ 55 nm emission.
- the fluorescence intensity drop of the BODIPY in 16-attached vesicles monitored in the confocal fluorescence mode (488 nm excitation and 535 ⁇ 25 nm emission) was much slower than in the 1-attached vesicles ( FIGS. 4 I-J ).
- the average fluorescence intensity drop from 20 selected vesicles was 9% ⁇ 20% (mean t standard deviation) for the first 15 minutes UV light exposure.
- compound 1 disturbs the lipid membrane upon UV-activation through a nanomechanical effect, which allows smaller BODIPY dye molecules to pass through the membrane.
- PC-3 has only been identified to cross contaminate other prostate adenocarcinoma cell lines but the source original PC-3 cell line has not been identified to be cross contaminated with any other cell line.
- F-12/DMEM Dulbecco's Modified Eagle Medium
- FBS fetal bovine serum
- Cells were grown in 75 cm 2 plastic culture flasks, with no prior surface treatment. Cultures were incubated at 37° C. 10% average humidity and 5% (v/v) CO 2 . Cells were harvested by treatment with 0.25% (v/v) trypsin solution for 5 minutes at 37° C. Cell suspensions were pelleted by centrifugation at 1000 rpm for 3 minutes, and were re-suspended in fresh medium by repeated aspiration with a sterile plastic pipette.
- F-12/DMEM Dulbecco's Modified Eagle Medium
- FBS fetal bovine serum
- Microscopy cells were seeded in untreated iBibi 100 ⁇ L live cell channels and allowed to grow to 40% to 60% confluence, at 37° C. in 5% CO 2 . At this stage, the medium was replaced and cells were treated with the studied nanomachines and co-stains as appropriate, with 0.1% DMSO (as detailed above) present in the final imaging medium. For live cell imaging. DMEM/F12 media (10% FBS) lacking phenol red was used from this point onwards. Following incubation, where Method B was used, the channels were washed with live cell imaging media and imaged using a purpose build incubator housing the microscope maintaining 37° C., 5% CO 2 and 10% humidity.
- All live cell imaging experiments used either NIH 3T3 mouse skin fibroblast cells, PC-3 cells, a grade 3 human prostate adenocarcinoma, or Chinese hamster ovary cells (CHO). These are all well-studied cell lines with discrete morphological compositions. Each molecular machine was used as a stock solution at 0.10 to 1.00 ⁇ M with total dimethyl sulfoxide (DMSO) concentrations not exceeding 0.1% in the final cell media in order to avoid unwanted cell membrane permeabilization, the DMSO being required for solubility of the organic nanomachines. All loading experiments are carried out in a light-suppressed manner and the possibility of induced or accelerated uptake due to interaction between the molecular motors and the applied co-stain has been eliminated using a series of individual and reversed loading experiments.
- DMSO dimethyl sulfoxide
- Cell toxicity was determined using a ChemoMetec A/S NucleoCounter 3000-Flexicyte instrument with Via1-cassette cell viability cartridge using the cell stain Acridine Orange for cell detection, and the nucleic acid stain DAPI for detecting non-viable cells and Annexin V for the detection of apoptosis.
- the optimized imaging parameters allow appropriately high scanning speed to follow natural homeostatic events and identify any induced morphological or fluorescent signal localization change. Meanwhile, they also allow sufficiently long integration time for each pixel so an adequate amount of photon signals can be collected.
- the pixel size is set as 1 ⁇ 5 of the laser spot size.
- the images were acquired using a bidirectional 2-line averaging sequence, which gives minimal dead time ( ⁇ 1 ms) between line scanning. The image size was adjusted to 100 ⁇ 100 ⁇ m in order to study 1 to 3 cells simultaneously. Each individual experiment was repeated 3 times on triplicate slides. On each slide, at least 5 well-separated areas were imaged (Table 2).
- the applied 100 Hz detection sequence was based on a bidirectional dual-channel continuous scanning method where a minimalistic non-damaging visible laser light (458 nm, 0.2 mW) is used in conjunction with the above detailed UV exposure. This is set as a 2 line/scan accumulation parallel acquisition sequence that is recorded as a function of time. Studied channels correspond to transmission images and UV-induced mitochondrial auto-fluorescence detected at 460 to 550 nm. Applicants further confirmed that the effectiveness of monitoring the UV-activated nanomechanical action on live cells using a conventional CW mercury-arc excitation source equipped with an epi-fluorescence setup consisting of a Zeiss Axiovert 200M inverted microscope, as discussed in FIGS. 18 A-C .
- the modular PhMoNa technique is based on a laser scanning confocal microscope (LSCM) harnessing spatially modulated illumination intensities, using an in situ generated raster-scanned standing wave excitation beam optical grid pattern.
- LSCM laser scanning confocal microscope
- Frame size was determined at 1024 ⁇ 1024 pixel, with ⁇ 2 digital magnification to ensure illumination flatness of field and 0.6 airy disc unit determining the applied pinhole diameter rendering on voxel to be corresponding to 62 ⁇ 62 nm 2 (frame size 125 ⁇ 125 ⁇ m 2 ) with a section thickness set at 188 nm (at 355 nm excitation).
- a HeNe or Ar ion laser was used to aid transmission image capture and when commercially available organelle-specific stains (e.g. MitotrackerRedTM or PI) were used to corroborate cellular compartmentalization or follow the onset of necrosis
- the threshold algorithm to control brightness automated by the Leica SP5 II software is calculated by the image specific signal-to-noise ratio or it is accessible post image-processing.
- PC-3 cells are also incubated for 30 minutes to 4 hours with 9 or 10, and then washed three times and further incubated for 24 hours in rotor free media to see if they retain their surface-bound molecular rotors. The cells are then UV-activated to see if necrosis would be induced at the previously established accelerated time points.
- Nanomachines 9 and 10 appear to detach from the cell-surface with their lack of concentration gradient from the media. Hence, there is no triggered apoptosis. There is no difference between 9 and 10 in these experiments ( FIGS. 12 A- 13 E ).
- control molecular motor-free NIH 3T3 cells and the cells loaded with either 9 or 10 are identical without any change in behavior or onset of UV-induced nanomechanical necrosis. This is the same as in overnight media incubation.
- the cells show signs of proliferation of 10 to 25%.
- NIH 3T3 cells incubated for 9 for 24 hours seem to be the same as the cells exposed for 1-2 hours with no change in rates upon UV-accelerated nanomechanical necrosis compared to motor-free blanks ( FIGS. 13 A-E ).
- NIH 3T3 cells incubated with 10 for 24 hours are similar to those incubated with 9 except that in the case of 10, the NIH 3T3 cells did not proliferate as much and upon UV irradiation. Rather, the cells die 15 to 20% faster with necrosis than untreated motor-free blanks ( FIGS. 14 A-E ).
- mono-peptide-bearing nanomachine 10 can be internalized or membrane bound upon 24 hours of incubation, but in smaller quantities that do not trigger any noticeable toxic effect that could lead to programmed apoptotic cell death, while 10 only accelerates necrosis with less than half the efficiency compared to identical experiments using the targeted PC-3 cells.
- the CHO cell line was studied in an identical manner through experiments described above using NIH 3T3 control cells.
- identical results were found as had been seen in the NIH 3T3 cells, confirming that nanomachines 9 and 10 selectively target the PC-3 cell line over CHO cells ( FIGS. 15 A-E ).
- the molecular machines preferably embed in the membranes to show opening because their mere presence in the medium may not be sufficient in all circumstances.
- a targeting addend that does not impede rotor operation. If using two relatively large addends, they might retard the rotor from interacting with the lipid bilayer, thereby slowing the nanomechanical perturbation of the membrane. Since these are molecular-sized, pore formation on the membranes is not immediate. The process can take about 1 minute to become detectable through leakage currents and twice that long based upon morphological changes. Sufficient rupture stress will have to be displayed by the rotors in order to be effective in bilayer disruption. Finally, shorter UV-actuation times of ⁇ 30 scan permit analytes in the medium to enter the cells before the cells can reach the stage of programmed cell death.
- the total dosage of UV illumination was on a similar level across the different experiments in this study. Accordingly, it is likely that the total dosage of UV illumination is an important parameter since Applicants observed the membrane rupture on similar time scales across the three experiments: synthetic vesicles, confocal imaging on three different live cells types, and whole patch clamp on a fourth live cell type.
- nanomechanical action can disrupt external or internal cellular membranes and it can be used to introduce analytes into cells or expedite cell death.
- the nanomachines can be tracked within a cell or used to target specific cells through unique cell-surface recognition elements.
- the efficacy of this method for in vitro studies was demonstrated. Extensions to in vivo applications can be envisioned, especially at locations where short UV-exposure is acceptable (e.g., dentistry, localized epidermis and colorectal treatments).
- the use of molecular motors that are activatable by two-photon-, near-infrared- or radio-frequency-inputs, would make broader in vivo treatments viable.
- Applicants demonstrate that a two-photon illumination in the near-infra red (IR) region can activate molecular motors 3, 8 and 9 (structures shown in FIGS. 2 C, 2 D- 1 , and 2 D- 2 ), thereby resulting in Propidium Iodide (PI) dyes entering NIH 3T3 or PC 3 cells.
- Two-photon microscopy studies of NIH 3T3 cells were executed on a Nikon E600 upright confocal microscope coupled to a tuneable (710-950 nm Coherent MaiTai) multiphoton source operating at (20%) 240 mW power using a 166 lps scan speed and 128 ⁇ 128 pixel frame resolution for MP-illumination.
- Images (512 ⁇ 512 pixel, 166 lps) were recorded using a 543 nm (1 mW HeNe) laser combined with a 560 nm long-pass filter to record PI (200 nM) fluorescence and follow the onset of induced necrosis.
- FIGS. 19 A-D show images of cells in live cell media (10% FBS) with no molecular motors present.
- FIG. 19 B shows images of identical cells but with a 1 ⁇ M control molecular motor 5 (structure shown in FIG. 2 C and described in Example 1 as lacking a rotor but still absorbing UV-light) using Method A (10 minute pre-incubation prior to imaging, as described in Example 1).
- FIG. 19 C shows images of identical cells but with 1 ⁇ M of molecular motor 3 present using Method A (10 minute pre-incubation prior to imaging, as described in Example 1).
- FIG. 19 D shows images of PC-3 cells with 1.0 ⁇ M of molecular motor 8 present using Method B (1 hour pre-incubation followed by washing and subsequent imaging, as described in Example 1).
- FIGS. 20 A-L show images of control NIH 3T3 cells in live cell media (10% FBS) with no molecular motors present.
- FIG. 20 B shows images of identical NIH 3T3 cells with 1 ⁇ M of molecular motor 5 present using Method A.
- FIG. 20 C shows images of identical NIH 3T3 cells with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 20 D shows images of NIH 3T3 cells with 1 ⁇ M of molecular motor 8 present using Method B (1 hour pre-incubation prior washing and subsequent imaging).
- FIG. 20 A shows images of control NIH 3T3 cells in live cell media (10% FBS) with no molecular motors present.
- FIG. 20 B shows images of identical NIH 3T3 cells with 1 ⁇ M of molecular motor 5 present using Method A.
- FIG. 20 C shows images of identical NIH 3T3 cells with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 20 D shows
- FIG. 20 E shows images of identical control PC3 cells with no molecular motors present.
- FIG. 20 F shows images of identical PC3 cells with 1 ⁇ M of molecular motor 3 present using Method A.
- FIG. 20 G shows images of identical PC3 cells with 1 ⁇ M of molecular motor 5 present using Method A.
- FIG. 20 H shows images of identical PC3 cells with 1 ⁇ M of molecular motor 3 present using Method B.
- FIG. 20 I shows images of identical PC3 cells with 1 ⁇ M of molecular motor 8 present using Method A.
- FIG. 20 J shows images of identical PC3 cells with 1 ⁇ M of molecular motor 9 present using Method A.
- FIG. 20 K shows images of identical PC3 cells with 1 ⁇ M of molecular motor 8 present using Method B.
- FIG. 20 L shows images of identical PC3 cells with 1 ⁇ M of molecular motor 9 present using Method B.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Embodiments of the present disclosure pertain to methods of opening a lipid bilayer by associating the lipid bilayer with a molecule that includes a moving component capable of moving (e.g., rotating) in response to an external stimulus; and exposing the molecule to an external stimulus before, during or after associating the molecule with the lipid bilayer. The exposing causes the moving component of the molecule to move and thereby open the lipid bilayer (e.g., by pore formation). The external stimuli may include an energy source, such as ultraviolet light. The opened lipid bilayer may be a component of cell membranes in vitro or in vivo. The opening of the lipid bilayer may allow for the passage of various materials (e.g., active agents, such as peptide-based drugs) through the lipid bilayer and into cells. Additional embodiments of the present disclosure pertain to the aforementioned molecules for opening lipid bilayers.
Description
- This application is a continuation of U.S. application Ser. No. 17/233,102, filed Apr. 16, 2021, which is a continuation of U.S. application Ser. No. 16/316,716, filed Jan. 10, 2019, now U.S. Pat. No. 11,154,623, which is a national phase application under 35 U.S.C. 371 of International Application No. PCT/US2017/042148, filed Jul. 14, 2017, which claims priority to U.S. Provisional Patent Application No. 62/362,206, filed Jul. 14, 2016, the entire contents of each of which are hereby incorporated by reference.
- Not applicable.
- Current methods of delivering materials through lipid bilayers suffer from numerous limitations, such as ineffective delivery due to cellular resistance, and lack of specificity for desired lipid bilayers. The present disclosure addresses the aforementioned limitations.
- In some embodiments, the present disclosure pertains to methods of opening a lipid bilayer by associating the lipid bilayer with a molecule that includes a moving component capable of moving (e.g., rotating) in response to an external stimulus; and exposing the molecule to an external stimulus. The exposing can occur before, during or after associating the molecule with the lipid bilayer. The exposing causes the moving component of the molecule to move. Thereafter, the movement facilitates the opening of the lipid bilayer. Additional embodiments of the present disclosure pertain to the aforementioned molecules.
- The molecules of the present disclosure may include various additional components. For instance, in some embodiments, the molecules of the present disclosure may also include a base component that is capable of embedding with a lipid bilayer. Additional components of the molecules of the present disclosure can also include, without limitation, targeting agents for directing the molecule to a desired lipid bilayer, tracing agents for tracking the molecule, solubilizing agents for maintaining the water solubility of the molecule, active agents that may be releasable from the molecule, and combinations thereof.
- In some embodiments, the lipid bilayer that is opened by the methods and molecules of the present disclosure is a component of cell membranes in vitro. In some embodiments, the lipid bilayer is a component of cell membranes in vivo.
- In some embodiments, the molecules of the present disclosure are associated with lipid bilayers in vivo in a subject. In some embodiments, the association step includes administering the molecule to the subject. In some embodiments, the administered molecules may be utilized to treat a disease in a subject, such as skin-related cancers (e.g., skin cancers, colorectal cancers, oral cancers, and vaginal cancers).
- The lipid bilayers of the present disclosure may be exposed to various external stimuli. In some embodiments, the external stimuli include an energy source, such as ultraviolet light, visible light, near-infra red light, a radio frequency energy source, a magnetic field, a two-photon energy source, an electric field, an electromagnetic field, and combinations thereof. In some embodiments, the energy source includes ultraviolet light.
- In some embodiments, the movement of the moving component of the molecules of the present disclosure facilitates the opening of the lipid bilayer by forming pores in the lipid bilayer. In some embodiments, the opening of the lipid bilayer allows for the passage of materials through the lipid bilayer. In some embodiments, the materials include, without limitation, analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof. In some embodiments, the opening of the lipid bilayer allows for the passage of materials through the lipid bilayer and into cells for various purposes.
-
FIG. 1 provides a scheme of a method of opening a lipid bilayer. -
FIGS. 2A-2D-2 provide illustrations of molecular motors and control molecules for disruption of lipid bilayers through molecular mechanical action.FIG. 2A provides a schematic of molecular machines atop a cell membrane that open the membrane by UV-activated nanomechanical action.FIG. 2B shows a representative molecular machine with a rotor portion that is light-activated to rotate relative to the larger bottom stator portion, where the addends (R) can be varied to provide the requisite solubility, fluorophores for tracking, or recognition sites for cellular targeting.FIG. 2C shows representative structures of various nanomachines, includingnanomachines nanomachines control compound 5 which has a stator segment but no rotor and no capability of being UV-activated, andcontrol compound 6 with a slow rotor.FIGS. 2D-1 and 2D-2 show representative structures of additional nanomachines, includingnanomachines nanomachines nanomachine 6 rotates at 1.8 revolutions per hour when activated with 355 to 365 nm light at 60° C., but only cis-trans isomerizes about the rotor-stator double bond at room temperature. -
FIG. 3 provides a schematic of dye releasing experiments upon UV illumination. -
FIGS. 4A-K show data related to UV light-activated molecular motor 1 (illustrated inFIG. 2C ) and its releasing of encapsulated dye molecules from synthetic bilipid vesicles through nanomechanical action.FIGS. 4A-F show data related to BODIPY dye release from the vesicles.FIG. 4A shows a dark-field image of the vesicles.FIG. 4B shows a fluorescence image ofmolecular motor 1.FIG. 4C shows fluorescence images of BODIPY dyes.FIG. 4D shows co-localization of the images inFIGS. 4A-C .FIG. 4E shows the fluorescence image of the BODIPY dye as a function of UV-exposure time.FIG. 4F shows normalized fluorescence intensity vs UV-exposure time of 20 vesicles from 6 different sets of movies. Since different vesicles have different initial intensities, all traces are normalized according to the first data point. The error bar stands for the standard deviation of the normalized intensities. The first data point does not have an error bar.FIGS. 4G-J show control experiments with compound 16 (illustrated inFIG. 4K ) instead ofmolecular motor 1.FIG. 4G shows a dark-field image of the vesicles.FIG. 4H shows a fluorescence image ofcompound 16. The image shows that 16 is incorporated into the lipid bilayer.FIG. 4I shows a fluorescence image of the BODIPY dye andcompound 16 as a function of UV-exposure time.FIG. 4J shows normalized fluorescence intensity vs UV-exposure time of 20 vesicles from 5 different sets of movies. The scale bar inFIG. 4A is 10 μm and is the same forFIGS. 4B-D .FIG. 4G andFIG. 4H . The scale bar inFIG. 4I is 2 μm and is the same for all the figures inFIG. 4E andFIG. 4I . -
FIGS. 5A-B show the optical properties ofmolecular machines FIG. 5A shows the UV/vis absorption and fluorescence spectra ofmolecular machine 1. Excitation was observed at 630 nm.FIG. 5B shows the UV/vis absorption and fluorescence spectra ofcompound 2. Excitation was observed at 474 nm. -
FIGS. 6A-D show images of NIH 3T3 cells in the presence of the fluorescentmolecular machines molecular machines FIG. 6A shows images of cells exposed tonanomachine 2, including a left image (green,C loading 500 nM/2 h, λex 514 nm, λem 520-540 nm, 2 mW); a middle image MitoTrackerRed (red,C loading 100 nM/30 min, λex 543 nm, λem 550-600 nm, 0.5 mW); and a right image that represents the two merged transmission images verifying mitochondrial localization.FIG. 6B shows images of cells exposed tonanomachine 1, including a left image (red,C loading 500 nM/1 h, λex 633 nm, λem 650-700 nm, 1 mW); a middle image LysoTrackerGreen (green,C loading 200 nM/5 min, λex 488 nm, λem 500-530 nm, 0.2 mW); and a right image that represents the two merged transmission images highlighting pit-like surface localization.FIG. 6C shows a merged transmission (488 nm, 0.2 mW) images demonstration time dependent 1 internalization. UV-activation has been achieved using parallel λex 355 nm, 20mW 400 nJ/voxel total dwell time for the corresponding times noted in the images.FIG. 6D shows fluorescent images demonstrating time-dependent dispersion of formed intracellular aggregates of 1 after a 1 hour incubation and wash cycles followed by UV-activation for the corresponding times noted in the images. All scale bars are 20 μm. -
FIGS. 7A-E show the effects ofnanomachines control molecule 5 on PC-3 cells upon UV-activation. The rate of necrotic cell death and permeabilization of analytes into the cells was recorded. The UV-exposure times are shown in each image.FIG. 7A shows blank cells without molecular motors.FIG. 7B shows cells exposed tonanomachine 3.FIG. 7C shows cells exposed tonanomachine 4.FIG. 7D shows cells exposed to controlmolecule 5. All the exposures occurred at 500 nM with 5 minute incubation before imaging.FIG. 7E shows an identical imagingsequence using nanomachine 3 with the introduction of 100 nM PI (red, λex 543 nm, λem 610-630 nm, 0.2 mW) confirming molecular mechanical cell permeabilization with intercalation of RNA and DNA primarily in the cell nuclei. All scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. -
FIGS. 8A-D show interactions ofcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of NIH 3T3 cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes at 500 nM at 5 minute incubation time. The UV-exposure times are shown in each image.FIG. 8A shows blank cells without molecular motors.FIG. 8B shows cells withcompound 3.FIG. 8C shows cells withcompound 4.FIG. 8D shows cells withcompound 5. All scale bars are at 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown combining: 4 to 6 individual microscope slides with 5 to 6 FOV on each with an average 2.1 to 2.7 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. Table 2 provides more details. -
FIGS. 9A-E show interactions of the cis-trans isomerizing 6 with NIH 3T3 and PC-3 showing no enhanced necrosis, and non-directional rotating demethylated 3 (Table 2) showing slowed necrosis. Recorded merged transmission (458 nm, 0.2 mW) and UV-induced mitochondrial auto-fluorescence (green, λex 355 nm, λem 460-550 nm, 20mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of NIH 3T3 and PC-3 cells depict time-dependent UV-activated cell morphological changes at 500 nM at 5 minutes incubation time using Method A. The UV-exposure times are shown in each image.FIG. 9A shows PC-3 cells withoutcompound 6.FIG. 9B shows NIH 3T3 cells withoutcompound 6.FIG. 9C shows NIH 3T3 cells withcompound 6.FIG. 9D shows PC-3 cells withcompound 6.FIG. 9E shows PC-3 cells with a demethylated version of compound 3 (no methyl group at the allylic position, so no unidirectional rotation). All scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 4 to 5 individual microscope slides with 21 to 25 FOV on each with an average 2.5 to 3.1 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 10A-E show the effects of UV-activatedcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of PC-3 cancer cells depict time dependent UV-activated nanomechanical-induced cell morphological changes. PI was added in all the experiments.FIG. 10A shows blank cells without molecular motors.FIG. 10B shows cells withcompound 7 before washing.FIG. 10C shows cells withcompound 8 before washing.FIG. 10D shows cells withcompound 7 after washing.FIG. 10E shows cells withcompound 8 after washing. Scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown combining 4 to 5 individual microscope slides with 5 to 7 FOV on each with an average 2.2 to 3.1 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 11A-F provide data indicating thatmolecular motors compounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of NIH 3T3 cancer cells depicting time dependent UV-activated nanomechanical-induced cell morphological changes. PI was added in all experiments.FIG. 11A shows cells withcompound 3.FIG. 11B shows blank cells without nanomachines.FIG. 11C shows cells withcompound 7 before washing.FIG. 11D shows cells withcompound 8 before washing.FIG. 11E shows cells withcompound 7 after washing.FIG. 11F shows cells withcompound 8 after washing. Scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 4 to 6 individual microscope slides with 5 to 7 FOV on each with an average 2.2 to 2.6 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 12A-E show the study of targeted nanomechanical action ofcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of PC-3 human prostate cancer cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes. PI was added to all the cell media. The UV-exposure times are shown in each image.FIG. 12A shows blank cells without molecular motors.FIG. 12B shows cells exposed tocompound 9 without washing.FIG. 12C shows cells exposed tocompound 9 followed by washing.FIG. 12D shows cells exposed to compound 10 without washing.FIG. 12E shows cells exposed to compound 10 followed by washing. All scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.1 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 13A-E show the study of nanomechanical action ofcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of cancer cells depicting time-dependent UV-activated nanomechanical-induced cell morphological changes. The UV-activation times are noted in each image and 100 nM PI was in the medium.FIG. 13A shows PC-3 blank cells without motors after 24 hours.FIG. 13B shows PC-3 cells exposed tocompound 9 and no washing after 24 hours.FIG. 13C shows PC-3 cells exposed tocompound 9, followed by washing, after 24 hours of incubation.FIG. 13D shows PC-3 cells exposed to compound 10 without washing after 24 hours.FIG. 13E shows NIH 3T3 cells exposed tocompound 9 without washing after 24 hours. Scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.1 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 14A-E show the study of the nanomechanical action ofcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of NIH 3T3 cells depicting time dependent UV-activated nanomechanical-induced cell morphological changes. The UV-activation times are noted in each image and 100 nM PI was in the medium.FIG. 14A shows blank cells without motors.FIG. 14B shows cells exposed tocompound 9 by a 1 hour incubation and no washing.FIG. 14C shows cells exposed tocompound 9 by a 1 hour incubation followed by washing.FIG. 14D shows cells exposed to compound 10 by a 1 hour incubation without washing.FIG. 14E shows cells exposed to compound 10 by a 1 hour incubation followed by washing. Scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 5 to 6 individual microscope slides with 4 to 6 FOV on each with an average 2.4 to 3.3 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 15A-E show the study of nanomechanical action ofcompounds mW 400 nJ/voxel total dwell time, 1024×1024 pixel) images of the study ofcompounds FIG. 15A shows blank cells.FIG. 15B shows cells exposed tocompound 9 without washing.FIG. 15C shows cells exposed to compound 10 without washing.FIG. 15D shows cells exposed tocompound 9 by 30 minutes of incubation with cells after washing.FIG. 15E shows cells exposed tocompound 9 with 24 hours of incubation followed by washing of cells. Scale bars are 20 μm. The statistical analyses for each of the live cell microscopy experiments are shown to the right of the images for that row. The determination of onset (orange) and final stage (red) of necrosis are shown by combining 3 to 5 individual microscope slides with 5 to 9 FOV on each with an average 2.5 to 3.2 cells per FOV. The displayed standard deviations are calculated from the Gaussian fit and have been rounded up to 15 second integers due to the experimentally predefined length associated with each scanning sequence. See Table 2 for more details. -
FIGS. 16A-B show whole-cell patch clamp studies of the dynamic effects of UV-induced molecular mechanical action ofcompound 3 upon HEK293 cells. Also shown are controlstudies using compound 3 without UV activation. UV-exposed rotor-free control molecule 5, and no molecular additives x.Compounds FIG. 16A shows transmembrane currents in HEK293 cells showing that cells treated with UV (355 nm)-activatedmolecular motors 3 have inward currents consistent with membrane degradation (bottom trace). Without UV illumination, cells treated withcompounds free compound 5, and no molecular additive x, show no inward currents during UV illumination (center two traces). Cells were held at −70 mV in voltage clamp mode and UV exposure began 15 seconds after the start of the recording. Each recording is a biological replicate and all traces are shown (n=4 recordings from different cells for 3+UV; n=3 recordings from different cells for each other condition).FIG. 16B shows representative differential interference chromatography (DIC) images of cells captured before (t=0) and after (t=4 min) exposure to UV in the presence ofcompounds compound 3. The scale bar represents 10 μm and is applicable for each micrograph. -
FIGS. 17A-D show the consecutive monitoring of the interconversion of cis and trans isomers ofcompound 8.FIG. 17A is a chromatogram of one of the isomers immediately after separation.FIG. 17B is a chromatogram of the fraction after being stored in the freezer for two weeks.FIG. 17C is a chromatogram of the fraction after being exposed to room light for 20 minutes.FIG. 17D is a chromatogram of the fraction after being irradiated with laboratory TLC UV light for 15 minutes. -
FIGS. 18A-C show data related to monitoring the UV-activated nanomechanical action ofcompound 3 upon exposure to NIH 3T3 cells using a conventional CW mercury-arc excitation source equipped with an epi-fluorescence setup consisting of a Zeiss Axiovert 200M inverted microscope. Shown are live cell microscopy images of a selected NIH 3T3 cell pre-incubated with 500 nm ofcompound 3 for 30 minutes in live cell imaging cell culture media (Method A), including; (1) (green) UV-induced mitochondrial autofluorescence (λex BP365/50 nm. λdm 395, λem LP420 nm, taq=880 ms/FOV); (2) (red) PI (100 nM) fluorescence (λex BP456/12 nm, λdm 570, λem LP580 nm, taq=370 ms/FOV); (3) (orange) RGB merge and (4) corresponding transmission images using a conventional epi-fluorescence setup and high resolution CCD camera. The selected FOV was exposed to continuous UV radiation (365/50 Band-pass filter) for an initial time (i.e., 0 minute) (FIG. 18A ), 5 minutes (FIG. 18B ), and 30 minutes (FIG. 18C ) to confirm UV-activated molecular-motor-induced cell necrosis. -
FIGS. 19A-D provide a demonstration that a two-photon illumination in the near-infra red (IR) region can activatemolecular motors FIG. 19A shows images of cells in live cell media with no molecular motors present.FIG. 19B shows images of identical cells but with a 1 μM controlmolecular motor 5 using Method A.FIG. 19C shows images of identical cells but with 1 μM ofmolecular motor 3 present using Method A.FIG. 19D shows images of PC-3 cells with 1.0 μM ofmolecular motor 8 present using Method B. The scale bars correspond to 20 μm. -
FIGS. 20A-L provide an additional demonstration that a two-photon illumination in the near-infra red (IR) region can activate themolecular motors FIG. 20A shows images of control NIH 3T3 cells in live cell media under two-photon illumination with no molecular motors present.FIG. 20B shows images of identical NIH 3T3 cells with 1 μM ofcontrol 5 present using Method A.FIG. 20C shows images of identical NIH 3T3 cells with 1 μM ofmolecular motor 3 present using Method A.FIG. 20D shows images of NIH 3T3 cells with 1 μM ofmolecular motor 8 present using Method B.FIG. 20E shows images ofidentical control PC 3 cells under two-photon illumination with no molecular motors present.FIG. 20F shows images ofidentical PC 3 cells with 1 μM ofmolecular motor 3 present using Method A.FIG. 20G shows images of identical PC-3 cells with 1 μM ofcontrol 5 present using Method A.FIG. 20H shows images of identical PC-3 cells with 1 μM ofmolecular motor 3 present using Method B.FIG. 20I shows images of identical PC-3 cells with 1 μM ofmolecular motor 8 present using Method A.FIG. 20J shows images of identical PC-3 cells with 1 μM ofmolecular motor 9 present using Method A.FIG. 20K shows images of identical PC3 cells with 1 μM ofmolecular motor 8 present using Method B.FIG. 20L shows images of identical PC-3 cells with 1 μM ofmolecular motor 9 present using Method B. The scale bars correspond to 20 μm. - It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and/or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that comprise more than one unit unless specifically stated otherwise.
- The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.
- An important aspect of biomedical therapy is the effective delivery of various molecules such as drugs and genetic information into cells. In order to be effective, such delivery methods must facilitate the passage of the molecules across the lipid bilayer of cell membranes. Thus, several physical techniques have been used to open lipid bilayers of cellular membranes. Such techniques use physical energies such as electric fields, magnetic fields, temperature, ultrasound, and light. These techniques have been used to intentionally introduce foreign materials into cells, release molecular species from cells, or to induce necrosis.
- Recently, molecular motors and switches that can change their conformation in a controlled manner after external stimulus have been explored to develop molecular machines that can have applications in the biomedical field. For example, the molecular photoswitch azobenzene has been adapted and used as a photochromic K+ channel opener, as an optical controller of insulin secretion, and for restoration of light sensitivity in blind retinae.
- However, current methods of facilitating the passage of materials through lipid bilayers continue to suffer from numerous limitations. For instance, many methods of delivering materials through lipid bilayers of cells become ineffective because cells develop resistance to the delivery agents. Furthermore, many of the existing methods of delivering materials into cells are not able to target desired lipid bilayers in a specific manner. The present disclosure addresses the aforementioned limitations.
- In some embodiments, the present disclosure pertains to methods of opening a lipid bilayer. In some embodiments illustrated in
FIG. 1 , the methods of the present disclosure include steps of associating the lipid bilayer with a molecule that includes a moving component capable of moving in response to an external stimulus (step 10), and exposing the molecule to an external stimulus (step 12). The exposure of the molecule to the external stimulus causes the moving component of the molecule to move (step 14). Thereafter, the movement of the moving component of the molecule facilitates the opening of the lipid bilayer (step 16). In some embodiments, the opening of the lipid bilayer can be used to facilitate the passage of various materials through the lipid bilayer (step 18). Additional embodiments of the present disclosure pertain to the aforementioned molecules for opening a lipid bilayer. - Examples of the methods and molecules of the present disclosure are also depicted in
FIG. 2A . In this depiction,molecule 22 with movingcomponent 24 becomes associated withlipid bilayer 20. Thereafter, the molecule is exposed to light irradiation (i.e., an external stimulus). The light irradiation causes movingcomponent 24 ofmolecule 22 to move (i.e., rotate in this embodiment) and facilitate the opening oflipid bilayer 20 through the formation of pores 26. - As set forth in more detail herein, the present disclosure can have numerous embodiments. In particular, various molecules may become associated with various types of lipid bilayers in various manners. Moreover, the molecules may be exposed to various external stimuli in order to open the lipid bilayers through various mechanisms. Furthermore, the molecules and methods of the present disclosure can be utilized to pass various materials through the opened lipid bilayers for various purposes and applications.
- Molecules for Opening Livid Bilayers
- The methods of the present disclosure can utilize various types of molecules for opening lipid bilayers. Additional embodiments of the present disclosure pertain to the aforementioned molecules for opening lipid bilayers.
- The molecules of the present disclosure generally include a moving component capable of moving in response to an external stimulus. In some embodiments, the moving components of the present disclosure can include one or more conjugated systems. In some embodiments, the wavelength of the conjugated system may shift to the visible region, thereby making the moving component activatable to visible light.
- In some embodiments, the molecules of the present disclosure also include a base component that is capable of embedding with a lipid bilayer. In some embodiments, the moving component is also capable of embedding with the lipid bilayer. In some embodiments, the base component is also capable of moving in response to an external stimulus.
- In some embodiments, the molecules of the present disclosure also include one or more targeting agents. In some embodiments, the targeting agent is capable of directing the molecule to a specific type of a lipid bilayer, such as a lipid bilayer associated with cell membranes of particular cells, organs, or tissues. In some embodiments, the targeting agent is capable of binding to a receptor on a lipid bilayer of a cell membrane.
- The molecules of the present disclosure may be associated with various types of targeting agents. For instance, in some embodiments, the targeting agent includes, without limitation, amino acids, peptides, proteins, aptamers, antibodies, small molecules, carbohydrates, polysaccharides, and combinations thereof. In some embodiments, the targeting agent includes peptides. In some embodiments, the targeting agent includes antibodies, such as monoclonal antibodies.
- In some embodiments, the targeting agents may be used to target specific cell types, such as cancer cells (e.g., cancer cells associated with a specific type of cancer, such as skin-related cancers), fat (adipocyte) cells, or diseased cells. In some embodiments, the targeted cells have an overexpressed and specific cell surface receptor that is recognized by the targeting agents.
- In some embodiments, the molecules of the present disclosure may also include one or more tracing agents. In some embodiments, the tracing agent can be utilized to track the association of molecules with a lipid bilayer.
- The molecules of the present disclosure may be associated with various types of tracing agents. In some embodiments, the tracing agents may be detectable by magnetic resonance imaging (MRI), positron emission tomography (PET), or other imaging techniques. In some embodiments, the tracing agents include, without limitation, fluorophores, chromophores, dyes, radio-labeled molecules, radioactive nuclei, high contrast agents, gadolinium, gallium, thallium, fluorinated compounds, and combinations thereof.
- In some embodiments, the molecules of the present disclosure also include one or more solubilizing agents. In some embodiments, the solubilizing agents help maintain the water solubility of the molecule.
- The molecules of the present disclosure may be associated with various types of solubilizing agents. For instance, in some embodiments, the solubilizing agents include, without limitation, peptides, glycols, alcohols, carboxylates, polysaccharides, salts, acids, polyethers, polyethylene glycols (PEGs), carbohydrates, and combinations thereof. In some embodiments, the solubilizing agents include glycols, such as polyethylene glycol units. In some embodiments, the solubilizing agents include alcohols, such as polyvinyl alcohol and polyols. In some embodiments, the solubilizing agents include carboxylates, such as carboxylate moieties. In some embodiments, the solubilizing agents include acids, such as sulfonic acids. In some embodiments, the solubilizing agents include salts, such as ammonium salts.
- In some embodiments, the molecules of the present disclosure may also include one or more active agents. In some embodiments, the molecules of the present disclosure may be associated with one or more active agents in a releasable manner. For instance, in some embodiments, the molecules of the present disclosure are releasably associated with one or more active agents through a cleavable bond, such as an ester linkage (e.g., cleavable by an esterase), an amide linkage (e.g., cleavable by an amidase), or a photolabile linkage (e.g., cleavable by UV light). In some embodiments, the molecules of the present disclosure are releasably associated with one or more active agents such that the one or more active agents are released from the molecules once the molecules facilitate the opening of the lipid bilayer or enter cells.
- The molecules of the present disclosure may be associated with various types of active agents. For instance, in some embodiments, the active agents include, without limitation, drugs, peptides, polypeptides, nucleotides. DNA, RNA, siRNA, enzymes, and combinations thereof. In some embodiments, the active agents include drugs, such as anti-cancer drugs. In some embodiments, the active agents include peptides. The use of additional active agents can also be envisioned.
- In more specific embodiments, the molecules of the present disclosure include the following structure (depicted as structure 1):
- Region A in
structure 1 includes moving component R3, which is capable of moving in response to an external stimulus. Region B instructure 1 includes a base component. In some embodiments, the base component is capable of embedding with a lipid bilayer. In some embodiments, the moving component is capable of embedding with a lipid bilayer. In some embodiments the moving and base components can embed with a lipid bilayer.Structure 1 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents. In some embodiments, the other components can also embed with the lipid bilayer. - R1 and R2 in
structure 1 can include various groups and moieties. For instance, in some embodiments. R1 and R2 can each independently include, without limitation, hydrogen, alkanes, alkenes, alkynes, carboxyl groups, ketone groups, alkoxy groups, methoxy groups, ethers, nitro groups, nitriles, sulfates, sulfonates, halogens, amine groups, amide groups, alcohols, aromatic groups, aryl groups, phenyl groups, annulated rings, carbohydrates, polysaccharides, peptides, targeting agents, tracing agents, fluorophores, solubilizing agents, active agents, and combinations thereof. - In some embodiments. R1 and R2 can each include annulated rings. In some embodiments, the annulated rings, when aromatic or further pi-electron-conjugated, can facilitate the activation of the molecules of the present disclosure by a lower energy source, such as visible light. X in
structure 1 can also include various groups and moieties. For instance, in some embodiments. X can include, without limitation, S, CH2, O, and combinations thereof. In some embodiments, X includes S. - Moving component R3 in structure 1 can also include various structures. For instance, in some embodiments, R3 includes the following structure (depicted as structure 2):
- In some embodiments. R3 in structure 1 includes the following structure (depicted as structure 3):
- The use of additional moving component structures can also be envisioned. For instance, in some embodiments, the moving components or molecules may have one or more annulated rings, such has annulated aromatic rings. Due to the additional annulated aromatic rings, the light absorbance and emission spectra of a molecule's moving component may undergo bathochromic shifts in some embodiments, thereby being excited in the visible region at 400 nm or higher wavelengths (e.g., at least 500 nm or even 700 nm).
- In some embodiments, the presence of additional annulated aromatic rings on a molecule or a moving component may facilitate the excitation of the moving component in the near infrared (IR) region at greater than 700 nm. In some embodiments, as excitation wavelength increases, the energy that the moving component exerts on the lipid bilayer decreases. Therefore, in some embodiments, an assessment of the energy requirements for lipid bilayer disruption followed by correlation to the excitation and emission energies of the molecule may be required. The Examples provide various methods for such energy calculation.
- In more specific embodiments, the molecules of the present disclosure include the following structure (depicted as structure 4):
- Region A in
structure 4 includes a moving component capable of moving in response to an external stimulus. Region B instructure 4 includes a base component that is capable of embedding with a lipid bilayer.Structure 4 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents. In addition. R1 and R2 can include various moieties and functional groups that were described previously. - In more specific embodiments, the molecules of the present disclosure include the following structure (depicted as structure 5):
- Region A in
structure 5 includes a moving component capable of moving in response to an external stimulus. Region B instructure 5 includes a base component that is capable of embedding with a lipid bilayer.Structure 5 can also include other components, such as targeting agents, tracing agents, fluorophores, solubilizing agents, and active agents. In addition, R1 and R2 can include various moieties and functional groups that were described previously. - The use of additional molecules capable of opening lipid bilayers can also be envisioned. For instance, more specific examples of the molecules of the present disclosure that resemble the molecules depicted in the aforementioned structures are shown in
FIGS. 2C-2D -2 (e.g., molecules 1-4 and 6-10). For the sake of clarity, aforementioned structures 1-5 are different from molecules 1-5 depicted inFIG. 2C . - Livid Bilayers
- The molecules and methods of the present disclosure can be utilized to open various types of lipid bilayers. In some embodiments, the lipid bilayers may be components of cell membranes, such as external or internal cellular membranes. In some embodiments, the lipid bilayers may be components of an organelle, such as the mitochondria. In some embodiments, the lipid bilayers may be components of a nuclear membrane.
- In some embodiments, the lipid bilayers may be components of cell membranes in vivo, such as cell membranes of a tissue or organ in a subject (e.g., a human being). In some embodiments, the cell membranes may be components of various cell types of interest, such as cancer cells, tumor cells, diseased cells, fat cells, and combinations thereof.
- In some embodiments, the lipid bilayers may be components of cell membranes in vitro, such as cell membranes in a cell culture medium. In some embodiments, the lipid bilayers may be components of vesicles, such as synthetic vesicles in vitro. The use of additional lipid bilayers can also be envisioned.
- Association of Molecules with Lipid Bilayers
- The molecules of the present disclosure may become associated with lipid bilayers in various manners. For instance, in some embodiments, the molecules of the present disclosure become embedded within the lipid bilayer. In some embodiments, the molecules of the present disclosure are inserted into the lipid bilayer. In some embodiments, the molecules of the present disclosure are placed on surfaces of the lipid bilayer.
- The molecules of the present disclosure may become associated with lipid bilayers by various steps. For instance, in some embodiments, the molecules of the present disclosure become associated with lipid bilayers by exposing the lipid bilayers to the molecules. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers by incubating the lipid bilayers with the molecules. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers by contacting the lipid bilayers with the molecules.
- In some embodiments, the molecules of the present disclosure become associated with lipid bilayers in vitro. In some embodiments, the molecules of the present disclosure become associated with lipid bilayers in vivo in a subject (e.g., a human being). In some embodiments, the molecules of the present disclosure become associated with lipid bilayers in vivo in a subject by administering the molecules to the subject.
- Various methods may be utilized to administer the molecules of the present disclosure to a subject. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, oral administration, inhalation, subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, intrathecal injection, topical administration, central administration, peripheral administration, and combinations thereof.
- In some embodiments, the molecules of the present disclosure may be co-administered to a subject with additional materials, such as active agents. In some embodiments, the administered molecules of the present disclosure may be releasably linked to an active agent. In some embodiments, the administered molecules may be utilized to treat a disease in a subject, such as skin-related cancers. In some embodiments, the skin-related cancers include, without limitation, skin cancer (e.g., melanoma), colorectal cancers, oral cancers, vaginal cancers, and combinations thereof.
- In more specific embodiments, the molecules of the present disclosure may be administered to a subject through intravenous administration (e.g., intravenous injection). In some embodiments, the molecules of the present disclosure may be administered onto a skin of subject through subcutaneous (i.e., subdermal) administration (e.g., subcutaneous injection). In some embodiments, the subdermal administration can occur in the presence of a skin-penetrating material, such as dimethylsulfoxide, thereby carrying the molecule through the skin. In some embodiments, the molecule (e.g., the molecule and its associated or co-administered active agent) can be carried through the skin using these dermal transport agents. In some embodiments, the dermal transport agents can also facilitate transport of the molecules of the present disclosure (e.g., the molecule and its associated or co-administered active agents) through the vaginal, colorectal, oral and gastrointestinal layers to facilitate transport of the molecules to their sites of interest before or during activation by exposure to an external stimulus (e.g., light or other external stimuli).
- In some embodiments, the molecules of the present disclosure may be co-administered to a subject along with an energy source capable of providing an external stimulus. In some embodiments, the energy source includes a light source, such as an LED lamp in capsule form. In some embodiments, the molecules of the present disclosure may be co-administered to a subject along with an energy source (e.g., LED lamp) and an active agent (e.g., peptide-based drug).
- In some embodiments, a capsule that contains an LED lamp and the molecules of the present disclosure (i.e., nanomachines) and an active agent (e.g., a drug associated with the molecule or co-administered with the molecule, such as a peptide-based drug) can be dissolved in the small intestines such that the LED lamp activates the molecules (i.e., nanomachines) which aid in opening the gastrointestinal lining to permit the active agent to enter the bloodstream. In some embodiments, the aforementioned administration can occur in conjunction with an epithelial transport agent. Additional administration methods can also be envisioned.
- The lipid bilayers of the present disclosure may be associated with the molecules of the present disclosure for various periods of time. For instance, in some embodiments, the association takes place from about 1 minute to about 48 hours. In some embodiments, the association takes place from about 5 minutes to about 48 hours. In some embodiments, the association takes place from about 5 minutes to about 2 hours.
- The lipid bilayers of the present disclosure may become associated with various concentrations of the molecules of the present disclosure. For instance, in some embodiments, the molecule concentrations range from about 10 nM to about 10 μM. In some embodiments, the molecule concentrations range from about 100 nM to about 1 μM. In some embodiments, the molecule concentrations range from about 100 nM to about 500 nM. In some embodiments, the molecule concentrations range from about 100 nM to about 200 nM. In some embodiments, the molecule concentrations are at least about 100 nM.
- Exposure of Molecules to External Stimuli
- The molecules of the present disclosure may be exposed to various external stimuli in order to cause the movement of their moving component. For instance, in some embodiments, the molecules of the present disclosure are exposed to an external stimulus that includes an energy source. In some embodiments, the energy source includes, without limitation, ultraviolet (UV) light, visible light, near-infra red (IR) light, a radio frequency (RF) energy source, a two-photon energy source, an electric field, a magnetic field, an electromagnetic field, and combinations thereof. In some embodiments, the energy source includes ultraviolet light. In some embodiments, the energy source is in the form of electromagnetic radiation.
- In some embodiments, a two-photon energy source is utilized to provide a very focused area of exposure. In some embodiments, the energy source includes two photons of near-infra red light. In some embodiments, the near-infra red light activates the molecules of the present disclosure by the use of two photons at about 710 nm.
- In some embodiments, the molecules of the present disclosure are exposed to an energy source at various wavelengths. For instance, in some embodiments, the wavelength of the energy source ranges from about 355 nm to about 365 nm. In some embodiments, the wavelength of the energy source ranges from about 500 nm to about 610 nm. In some embodiments, the wavelength of the energy source ranges from about 600 nm to about 750 nm. Additional wavelength ranges can also be envisioned.
- The molecules of the present disclosure may be exposed to an external stimulus for various periods of time. For instance, in some embodiments, the exposure time may be from about 1 second to about 600 seconds. In some embodiments, the exposure time may be from about 1 second to about 400 seconds. In some embodiments, the exposure time may be from about 1 second to about 300 seconds. In some embodiments, the exposure time may be from about 1 second to about 200 seconds. In some embodiments, the exposure time may be from about 1 second to about 60 seconds. In some embodiments, the exposure time may be from about 1 second to about 30 seconds. Additional exposure times can also be envisioned.
- The molecules of the present disclosure may be exposed to an external stimulus at various periods of time. For instance, in some embodiments, the exposing occurs after the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs before the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs while the molecule is associated with a lipid bilayer. In some embodiments, the exposing occurs before or during the association of the molecule with the lipid bilayer.
- The molecules of the present disclosure may be exposed to external stimuli in various environments. For instance, in some embodiments, the molecules of the present disclosure are exposed to an external stimulus in vitro. In some embodiments, the in vitro environment may be a cell culture medium that contains lipid bilayers as components of cell membranes.
- In some embodiments, the molecules of the present disclosure are exposed to an external stimulus in vivo. In some embodiments, the in vivo environment may be the organ or tissue of a subject that has been administered with the molecules of the present disclosure. In some embodiments, the in vivo environment may be the skin of a subject that has been administered with the molecules of the present disclosure.
- Molecular Movement
- The exposure of the molecules of the present disclosure to an external stimulus can have various effects on the molecules. In particular, the exposure causes the moving component of the molecule to move in various manners in response to the external stimulus. For instance, in some embodiments, the movement includes, without limitation, rotation, flapping, jumping, and combinations thereof. In some embodiments, the movement includes flapping.
- In some embodiments, the movement is confined to the moving component of the molecule. In some embodiments, the movement occurs throughout the entire molecule.
- In more specific embodiments, the movement includes rotation. The moving components of the molecules of the present disclosure can rotate in various manners. For instance, in some embodiments, the moving component of the molecules of the present disclosure rotates in a unidirectional manner. In some embodiments, the moving component rotates in a non-reciprocating unidirectional manner. In some embodiments, the moving component rotates in a bi-direction manner. In some embodiments, the moving component rotates relative to a base component of the molecule.
- The moving component of the molecules of the present disclosure can rotate for various degrees in response to an external stimulus. For instance, in some embodiments, the moving component rotates from about 45 degrees to about 360 degrees. In some embodiments, the moving component rotates from about 60 degrees to about 180 degrees. In some embodiments, the moving component rotates for at least about 180 degrees. In some embodiments, the moving component rotates for at least about 360 degrees.
- The moving component of the molecules of the present disclosure can also rotate at various rates in response to an external stimulus. For instance, in some embodiments, the moving component of the molecules of the present disclosure can rotate at rotation rates of about 1-10 MHz. In some embodiments, the moving component of the molecules of the present disclosure can rotate at rotation rates of about 2-3 MHz.
- In some embodiments, the moving component of the molecules of the present disclosure can rotate at 1-10 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at 1-5 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at 1-2 revolutions per hour. In some embodiments, the moving component of the molecules of the present disclosure can rotate at about 1.8 revolutions per hour.
- Opening of Livid Bilayers
- The movement of the moving component of the molecules of the present disclosure can have various effects on lipid bilayers. In some embodiments, the movement facilitates the opening of the lipid bilayers in various manners.
- For instance, in some embodiments, the movement of the moving component of the molecules of the present disclosure changes the conformation of the molecule (e.g., change in the conformation of
molecule 22, as depicted inFIG. 2A ). Thereafter, the change in the conformation of the molecule leads to the opening of the lipid bilayer. In some embodiments, the movement produces a tangential mechanical force that leads to the opening of the lipid bilayer. - In some embodiments, the movement of the moving component of the molecules of the present disclosure facilitates the opening of lipid bilayers by disrupting the lipid bilayers. In some embodiments, the movement facilitates the opening of the lipid bilayers by dislocation of lipid bilayer molecules. In some embodiments, the movement facilitates the opening of the lipid bilayers by causing rupture or degradation of the lipid bilayers.
- The opening of lipid bilayers by the molecules of the present disclosure may result in the formation of various structures. For instance, in some embodiments, the lipid bilayers are opened by forming pores (e.g., pores 26, as depicted in
FIG. 2A ). In some embodiments, the formed pores may be transient. In some embodiments, the formed pores may be permanent. In some embodiments, the formed pores may have various diameters. In some embodiments, the pore diameters range from about 10 nm to about 500 μm. - Applications and Advantages
- The methods and molecules of the present disclosure may be utilized to open various types of lipid bilayers for various applications. For instance, in some embodiments, the methods and molecules of the present disclosure can be utilized to open lipid bilayers for passage of various materials through the lipid bilayer. In some embodiments, such materials can include, without limitation, analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof. In some embodiments, the materials can include nucleotides, such as siRNA, DNA, RNA, and combinations thereof.
- In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into cells. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and out of cells. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into vesicles. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and out of vesicles.
- In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into organelles of cells, such as the mitochondria. In some embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into the nucleus of cells.
- In more specific embodiments, the methods and molecules of the present disclosure can be utilized for passage of materials through a lipid bilayer and into cells in order to induce various effects on the cells. In some embodiments, such induced effects can include, without limitation, cell death, necrosis, disease treatment, and combinations thereof. In some embodiments, such effects can occur in vitro (e.g., in a cell culture medium) or in vivo (e.g., in a subject).
- In more specific embodiments, the methods and molecules of the present disclosure can be utilized to open lipid bilayers of a cell membrane in a cell culture medium. Such methods can include associating the lipid bilayers of the cell membranes with the molecules of the present disclosure and exposing the molecules to an external stimulus in the presence of various materials (e.g., analytes, active agents, drugs, nucleotides, DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof) such that the materials enter the cells after exposure and induce various effects on the cells (e.g., cell death, necrosis, disease treatment, and combinations thereof).
- In further embodiments, the methods and molecules of the present disclosure can be utilized to open lipid bilayers of a cell membrane associated with an organ or a tissue in a subject by administering the molecules of the present disclosure to the subject such that the lipid bilayers of the cell membranes of the desired organ or tissue become associated with the molecules of the present disclosure. The desired organ or tissue may be exposed to an external stimulus in the presence of various co-administered materials (e.g., analytes, active agents, drugs, nucleotides. DNA, RNA, siRNA, polypeptides, enzymes, polysaccharides, imaging agents, and combinations thereof) such that the materials enter the cells of the tissue or organ after exposure and induce various effects (e.g., cell death, necrosis, disease treatment, and combinations thereof).
- In some embodiments, the methods and molecules of the present disclosure can be utilized for the sculpting of cells, such as fat (adipocyte) cells. For instance, in some embodiments, the molecules of the present disclosure may be administered (e.g., subcutaneously injected) under the skin of a subject where fat cells reside (or administered through other methods, such as transdermal administration or intravenous injection). An external stimulus (e.g., light or other external stimuli, such as radiofrequency fields, electric fields or magnetic fields) may be applied outside the skin to expose the molecules of the present disclosure to irradiation (e.g., UV, visible (e.g., red light) or two-photon near-infra red irradiation) through the skin and necrose the fat cells. After a few days, the aforementioned steps may be repeated for additional sculpting. In some embodiments, the sculpting of cells in accordance with the methods of the present disclosure can be utilized to selectively remove cells (e.g., fat cells) from a desired area.
- In additional embodiments, the methods and molecules of the present disclosure can be utilized to treat skin-related cancers, such as skin cancer (e.g., melanoma), colorectal cancers, oral cancers, and vaginal cancers. In some embodiments, the molecules of the present disclosure are administered to the affected cells (e.g., via subcutaneous injection or oral administration). An energy source (e.g., a light source, such as a light source via an endoscope or an administered pill) could then be utilized to expose the molecules to an external stimulus (e.g., UV irradiation) and destroy those cells. In some embodiments, a UV light source (e.g., at wavelengths of about 350-360 nm) or a near-infra red light source could be utilized to irradiate the cells.
- In more specific embodiments, the methods and molecules of the present disclosure can be utilized to facilitate the uptake of various active agents (e.g., peptide-based drugs) by a subject. For instance, in some embodiments, the active agent may be co-administered to a subject along with the molecules of the present disclosure and an energy source that is capable of providing an external stimulus (e.g., an LED light). In some embodiments, the molecules of the present disclosure may be associated with the active agents and co-administered with the energy source. In some embodiments, the co-administration occurs through the use of a carrier, such as a capsule. In some embodiments, the carrier may release the active agent (e.g., peptide-based drug), the molecule (i.e., the nanomachine), and the energy source (e.g., an LED light) near a particular cellular region, tissue or organ of the subject (e.g., the jejunum). Thereafter, the molecule associates with lipid bilayers and is activated by the energy source. This in turn facilitates the lipid bilayers of the particular cellular region, tissue or organ to open and uptake the active agents.
- Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicants note that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.
- In this Example, Applicants use nanomechanical action to open cellular membranes by association of molecular motors with lipid bilayers, and then activating the motors with light. Using precisely designed molecular motors and complementary experimental protocols, nanomechanical action can (a) induce the diffusion of analytes out of synthetic vesicles, (b) enhance diffusion of traceable molecular machines into and within live cells, (c) induce necrosis, (d) introduce analytes into live cells, and (e) be selectively targeted to specific live cell-surface recognition sites through nanomachines bearing short peptide addends. Applicants demonstrate that, beyond in vitro applications demonstrated in this Example, in vivo use can follow, especially through the use of two-photon-, near-infrared- and radio-frequency-activated domains.
- A scheme for nanomechanical action upon a lipid bilayer is shown in
FIG. 2A and the general design of a molecular machine suitable for transport though a lipid bilayer is shown inFIG. 2B . As seen inFIG. 2C , these include molecular motors bearing fluorophores for tracking (1 and 2), smaller nanomachines (3 and 4), a control that bears a stator but no rotor (5), a control analogue (6) that can only undergo cis-trans isomerization (flapping) at room temperature, and targeting systems that bear peptide sequences for binding to specific cell-surface receptors (7-10). - Applicants previously demonstrated that
molecular machine 1 displays enhanced diffusion in solution when the fast light-driven motor is activated by 355-365 nm UV light. - Applicants envision that similar motor-bearing nanomachines could be activated while associated with lipid membranes.
- First, synthetic bilipid vesicles were opened using
nanomachine 1 to release BODIPY dyes that were co-encapsulated with 1 in the vesicle (FIG. 3 ). The release of BODIPY dye molecules (not nanomachine-bound) encapsulated in a bilipid vesicle was studied with UV-exposure, and there was little release of the BODIPY dye from the synthetic vesicle. - Next, BODIPY and 1 were co-encapsulated in the bilipid vesicles, and a UV light-emitting diode was used as the activation source for 1 (
FIGS. 4A-K ). As the UV irradiation time increased, the fluorescence intensity of the vesicles declined as BODIPY and 1 diffused out of the vesicles, suggesting nanomechanical disruption of the vesicle bilipid membranes. A series of control molecules were used to exclude the possibility that the large fluorescence intensity drop in the vesicles containing the mixture of BODIPY and 1 was caused by the UV light induced photo-bleaching. The thermal effects due to the absorption of UV light by 1 was not responsible for the vesicle opening since a control molecule that has an even larger absorption coefficient at 365 nm than that of 1 did not show the loss of BODIPY fluorescence from the vesicles (FIGS. 4A-K ). - Following the synthetic bilipid vesicle experiments, nanomechanical action upon live cells was studied using confocal microscopy aided by a super-resolution technique called Phase Modulation Nanoscopy. Applicants used two experimental methods. In the first method (Method A), the molecular motors were loaded into the cell media and the imaging was initiated within 5 minutes to 24 hours. In the second method, (Method B), the molecular motors were loaded into the cell media, incubated for 30 minutes to 24 hours, and then washed three times with fresh molecular motor-free media before imaging.
- Applicants first studied the effect of the nanomachines on the cells without UV-light exposure. Using Method A, molecular machines 1 (λex 633 nm, λem 650-680 nm for the pendant cy5 dyes) and 2 (λex 514 nm, λem 530-580 nm for the pendant BODIPY dyes) do not induce accelerated necrosis when introduced to NIH 3T3 cells. However, due to 1 and 2 having well-pronounced visible fluorescence properties corresponding to the cy5 and BODIPY addends (
FIGS. 5A-B and Table 1, respectively), their intracellular uptake, motion or protein/organelle-assisted trafficking are clearly observed. The two luminescent compounds display very different localization patterns.Nanomachine 2 enters the cell and localizes in the mitochondria (FIG. 6A ). Conversely,nanomachine 1, when introduced to cells, displays pit-like cell surface localization (FIG. 6B ) and later, at 4 hour, small ˜1 μm aggregates are seen inside the cytoplasm. -
TABLE 1 Optical properties of molecules Compound λabs(nm) λem (nm) Φ f1 656 680 0.30a 2 507 518 0.75b aQuantum yield was measured using zinc phthalocyanine as a reference, (Φf = 0.30 in benzene, λex = 630 nm). bQuantum yield was measured using rhodamine 6G as a reference, (Φf = 0.94 in ethanol, λex = 474 nm). - These respective time-dependent localization patterns were observed to be constant within the applied 0.10-1.00 μM final nanomachine loading concentrations, suggestive of an active uptake mechanism. Applicants undertook a series of control experiments to confirm active molecular motor uptake and suspected endocytosis, using a range of nanomachine loading concentrations (0.10-1.00 μM. Method A) and incubation times of 5-60 minutes at 4° C., which is a temperature of general endocytosis inhibition.
- The aforementioned studies reveal that there was no detectable localized fluorescence of 2 in the mitochondria or 1 on the cell surface. These experiments eliminate the possibility of passive concentration gradient-driven diffusion or static cell surface interactions of these nanomachines. Further strengthening this observation are the fluorescence intensity measurements where more than 99% of motors applied could be recycled from the wash solutions and re-collected during loading and imaging. Since the motors were not UV-activated.
- Applicants did not see accelerated cell death. Cell viability throughout these experiments remained at >90% and both
fluorescent nanomachines - The NIH 3T3 cells in the presence of the nanomachines were then studied with concomitant UV activation. Upon UV-induced motor activation for 150 seconds (355 nm), 1, introduced by Method A, was found to cross the cell membrane, and it was internalized into cells in a time-dependent manner, displaying fast accelerated intracellular motion, compared to natural homeostatic cellular organelle movement in the absence of UV-nanomechanical activation (
FIG. 6C ). Combined controlled time and UV-exposure-dependent experiments indicate that the small aggregates of 1 inside the cytoplasm dissolve or burst with further increasing of fluorescence signal in the cytoplasm (FIG. 6D ). Thus, nanomachine trafficking can be facilitated and precisely observed. - Next, Applicants used the smaller nanomachine, 3. Initial control experiments (blank) were performed without nanomachines being present. UV-induced (355 nm) PC-3 necrosis is not initiated until approximately scan 20 (corresponding to 300 second continuous scanning UV exposure, 400 nJ/voxel total dwell time), characterized by massive disruption and rupture of the mitochondrial network and subsequent well-pronounced auto-fluorescent signal detectable in the nucleoli and nucleus wall (
FIG. 7A ). This is observable by the nucleoli membrane permeabilization and DNA damage at the onset of necrosis. The UV-induced necrosis reaches final stages at approximately scan 40 (corresponding to 600 seconds) that is consistent with characteristic extracellular membrane rupture and homogenous cytosolic auto-fluorescence, indicating loss of cellular organelle boundaries. - Conversely, visual signs of apoptosis involve cell shriveling and subsequent detachment from the cover-slip surface followed by fragmentation. Using 3 by Method A with both PC-3 and NIH 3T3 cell lines in the time- and UV-exposure-dependent in vitro microscopy experiments with the previously established standard experimental parameters and 355 nm laser exposure, >50% accelerated cell death (relative to UV-exposure without 3) is observed due to disruption of the cell membrane (
FIG. 7B and FIGS. SA-D). - Well-pronounced fragmentation of the mitochondrial network is established between
scan 8 and 10 (corresponding to 120 to 150 s, respectively) with extracellular membrane burst manifesting at scan 20 (300 seconds) (Table 2 andFIGS. 8A-D ). This is confirmation of accelerated necrosis. Importantly, 3 was found to be non-internalizing prior to UV-activation. Onset of necrosis and internal cell organelle mitochondrial fragmentation or nucleolus damage is identical regardless of prior 24 hour incubation of the cells with 3 vs. adding 3 directly before UV-activation. -
TABLE 2 Data sets for time of fluorescent nanomachine cell incorporation and for necrosis determinations. Avg. Time of Time of % No. of No. of Initial Full acceleration Std. Cell FOVb/ Cells/ Necrosisd Necrosis for full dev. Compound Type Methoda slide FOV NTotal c (s) (s) necrosise (s)f Blank NIH 3T3 A 25/5 2.4 60 300 600 0 15 Blank PC-3 A 25/5 3.1 78 300 600 0 30 Blank CHO A 25/3 2.5 64 300 600 0 30 1 NIH 3T3 A 27/5 2.6 70 300 600 0 15 2 NIH 3T3 A 28/5 2.3 65 300 585 2.5 30 1 NIH 3T3 B 25/5 2.6 65 300 600 0 30 2 NIH 3T3 B 26/5 2.7 70 300 570 5 30 3 NIH 3T3 A 30/6 2.2 66 150 300 50 30 3 PC-3 A 28/6 2.7 76 135 300 50 30 4 NIH 3T3 A 32/5 2.1 67 270 540 10 15 4 PC-3 A 25/5 2.5 63 270 540 10 15 5 NIH 3T3 A 22/4 2.7 60 300 600 0 15 5 PC-3 A 25/4 2.4 60 300 600 0 15 6 NIH 3T3 A 21/4 2.8 58 300 615 0 30 6 PC-3 A 24/4 2.5 60 300 600 0 30 7 NIH 3T3 A 27/4 2.4 65 150 390 40 30 7 PC-3 A 24/4 2.8 67 165 360 35 30 8 NIH 3T3 A 23/5 2.6 60 150 375 37.5 30 8 PC-3 A 29/4 2.2 64 150 360 40 15 7 NIH 3T3 B 32/5 2.0 64 165 390 40 30 7 PC-3 B 25/5 2.6 65 180 390 35 30 8 NIH 3T3 B 27/5 2.3 62 150 360 40 30 8 PC-3 B 28/5 2.4 67 150 360 40 30 9 NIH 3T3 A 24/5 2.7 65 300 570 5 30 9 PC-3 A 25/5 3.0 75 150 360 40 30 9 CHO A 23/5 3.1 71 300 585 2.5 30 10 NIH 3T3 A 27/6 2.6 70 270 540 10 30 10 PC-3 A 27/5 2.7 73 195 420 30 30 10 CHO A 24/5 2.9 72 270 540 10 30 9 NIH 3T3 B 25/5 2.8 70 300 600 0 15 9 PC-3 B 32/6 2.3 74 180 390 35 30 9 CHO B 26/4 2.8 72 285 570 5 30 10 NIH 3T3 B 22/5 3.3 73 300 600 0 30 10 PC-3 B 31/6 2.5 78 210 435 27.5 30 10 CHO B 22/4 3.2 71 300 600 0 15 NonMe-3 PC-3 A 27/5 2.4 65 270 540 10 30 aNanomachine loading method used before imaging. bFOV = field of view. cEach individual statistical analysis have been executed on N number of total cells per corresponding experiment (no data was ever excluded and all images were used without post-processing) according to a standard Gaussian (2-tailed) fit. dReported are the averaged time of death. eThe percentage is in acceleration. For example, 50% means cells die 50% faster when rotors are present with UV exposure verses UV upon blank cells, the latter meaning that no nanomachines are present. Zero percent means blank reference time or no observed acceleration in necrosis time. fStandard deviation is calculated for time of full necrosis where the minimum precision and experimental error, due to the nature of microscopic imaging sequence, is 15 s. No difference has been found between the standard deviation for initial or full time of necrosis within this experimental error. Initial necrosis or the onset of necrosis is determined when the mitochondrial network of the cell in the green channel, using mitochondrial autofluorescence, is dispersed evenly in the cytosol and the small nucleoli in the nucleus starts to become visible with higher than 10/255 contrast value. Full necrosis is determined when the cell membrane bursts showing observable lesions (cell organelle-free clear blebbing) on the cell membrane, and when PI is used the PI can be detected in nucleus at >10/255 contrast value. All statistical analyses have been undertaken using Origin2015 fitting to a Gaussian fit function y = y0 + a/(w*sqrt(pi/(4*ln(2)))) * exp(−4*ln(2)*(x − xc)2/w2) using the Levenberg-Marquardt iteration algorithm. All standard deviations have been established from each individual fitting and presented on the corresponding relevant graph. These values are also tabulated alongside relevant descriptive statistical parameters and rounded up to the nearest 15 seconds. -
Molecular machine 4 was studied using identical pre-set study parameters and Method A on both PC-3 and NIH 3T3 cells, where UV-induced nanomechanical action caused necrosis only 10% earlier (Table 2) than the standard blank non-molecular motor containing reference cells (FIGS. 7C andFIGS. 8A-D ).Nanomachine 4, bearing the larger aryl sulfonate moieties, might have been inhibited from having its rotor interact well with the cell membranes, or the addends themselves sterically encumbered their rotation near the membrane. - The rotor-
free control molecule 5 was studied to ensure that the rotary action is preferred for the bilayer perturbations. Using Method A on both PC-3 and NIH 3T3 cells, which has the same homopropargylic alcohol stator moieties as does 3,control molecule 5 shows no effect on necrosis upon standardized UV-exposure (FIGS. 7D andFIGS. 8A-D ). This further suggests that the accelerated cell death seen with 3 was not primarily due to the short exposure to UV light or subsequent thermal processes, similar to Applicants' earlier synthetic bilayer studies. - In order to further ensure that a fast rotary motion was preferred for nanomechanical opening of cells, another control (6) was used, which bears a 6-membered heterocyclic rotor that is nearly identical in molecular size and functionality to 3. However, 6 can only undergo cis-trans isomerization upon light activation at room temperature. This “flapping” action will occur without full rotation since that barrier (rotor crossing over the stator) requires 60° C. in the heterocyclic system. Even at 60° C., the rotation rate is only ˜2 revolutions·h−1 as opposed to the nearly identical molecular sized 3, which rotates at 2-3 MHz upon UV-activation at room temperature.
-
Compound 6 showed no enhanced necrosis in PC-3 or NIH 3T3 cells upon standardized UV-exposure (FIGS. 9A-E ). Furthermore, a compound analogous to 3 but without the allylic methyl, which therefore can rotate but not unidirectionally, was only slightly faster than the motor-free system (FIGS. 9A-E ). Therefore, the non-reciprocating unidirectional motor rotation is the highly preferred mode for these nanomachines that bear ultra-low Reynolds numbers, while progressing through the lipid bilayers. - Applicants further confirmed the nanomechanical opening and subsequent permeabilization of the membrane by adding a dye to the cell medium to assess its exogenous entry into the cells that might be afforded by nanomechanical action. Using PC-3 cells and 3, propidium iodide (PI, total concentration 0.10 μM) was introduced to the cell medium immediately prior to the time-dependent standardized imaging sequence. PI is a fluorescent intercalating agent that is not internalized by healthy cells, and it is non-toxic as shown by Applicants' molecular machine-free controls. Upon membrane disruption by nanomechanical action of 3 (
FIG. 7E ). PI enters the cell, travels to RNA- and DNA-rich areas where it intercalates and its excitation maximum subsequently displays ˜30 nm bathochromic shift (from 535 to 565 nm) accompanied by a parallel hypsochromic emission maximum shift (from 617 nm to 600 nm). - Internalized RNA- and DNA-induced PI fluorescence is detected between 600 and 630 nm, allowing time-dependent light-activated molecular motor-induced cell permeabilization to be confirmed. Further, PI was used to follow membrane damage that is due to UV-activated nanomachine activity leading to necrosis. Since the entry of the PI is on a relatively short time scale compared to the time of cell division, the cell has insufficient time to adopt programmed cell death (apoptosis). This was confirmed using Annexin V, an apoptosis-specific stain where no relevant fluorescence from this dye was observed throughout the course of the experiments.
- Considering the above UV-induced nanomechanical action, the peptide-bearing structures (7-10) were investigated to target specific cells for nanomachine-activated necrosis. The targeted cell line was PC-3 while NIH 3T3 and CHO cells were used as non-targeted controls. No selectivity was observed with the shorter
peptide targeting moieties 7 and 8 (Table 2 andFIGS. 10A-11F ), but the longer peptide sequences provided by 9 and 10 showed that the targeted PC-3 cells started to die of UV-activated motor-induced necrosis at 150 to 180 s, which corresponds to 40-50% faster onset than the molecular-motor-free UV-exposed cells or the untargeted molecular motor/cell combinations with NIH 3T3 and CHO cells (Table 2 andFIGS. 12A-15E ). - Several notable features became apparent in nanomachine design. Pre-binding to or insertion into to the cell membrane is preferred. Just being present in the medium will not result in accelerated rotor-induced UV activated necrosis. With 3, all cell types showed accelerated activated necrosis presumably because the core on 3, with its smaller addends, interacted well with the membranes and it had minimal intermolecular steric interference while transporting through the membranes. The mono-addended core of 10 is better able to approach the membrane to sufficiently close proximity than the more sterically hindered 9, but still not as efficiently as the smaller 3. Finally, better transport through the membrane was realized with the less sterically encumbered 10 over 9. Doubling the large addends could sterically slow the membrane-transport.
- The dynamic effects of nanomechanical action upon cellular membranes were then studied through the whole cell patch clamp electrophysiology of human embryonic kidney 293 cells (HEK293) commonly used for electrophysiological interrogation. Using Method B, the studies reveal that upon UV (355 nm) activation of
molecular motor 3, inward ionic currents were produced consistent with hydrophilic pores forming in the cellular membranes. These inward currents were not observed in the absence of UV illumination or during UV illumination of non-rotor-bearingcontrol 5 or UV illumination of untreated cells (FIG. 16A ). - Inward currents produced during UV illumination of cells treated with 3 then continued even in the absence of UV illumination suggested that the cell membranes were irreversibly damaged. This was accompanied by induced morphological changes to the cells, such as membrane blebbing (
FIG. 16B , white arrows), cell swelling and cytoplasmic degradation, all indicative of cell death. Although membrane blebbing occurs during apoptosis and necrosis, the large diameter of the blebs observed here (FIG. 16B ; r=3.8±0.2 μm) matches necrosis as does the observed cell-swelling and the absence of apoptotic bodies. Consistent with Applicants' previously observed delayed morphological effects on the other cell lines studied above, inward currents in HEK293 cells appeared between 40 and 60 seconds after exposure to UV illumination. The slow rise in inward current during illumination suggests an accumulation of many small pores or increasing pore sizes. - Membrane rupture and pore formation under a tangential mechanical force have been studied theoretically and experimentally. Specific to the nanomechanical forces in Applicants' experiments, the actuation of the rotor will produce a tangential mechanical force perturbing the membrane structure. The UV photon energy (λ=365 nm) that actuates the motor is E=hc/λ=5.4×10−19 J.
- If the entire amount of energy is used for the force generation, and the linear moving distance of the tip of the rotor is on the magnitude of s=1 nm, the generated force would be F=E/s=0.54 nN. The stress applied on the membrane would be 540 mN m−1, far exceeding the requisite rupture stress for most bilipid membranes of 1-30 mN m−1.
- Even if Applicants consider that the nanomechanical action is pulsed and the membrane is more resistant to rupture, it is still theoretically sufficient to disrupt the membrane locally and to eventually compromise its integrity. This conclusion is also consistent with the energetics estimation. The estimated free energy for pore formation is tens of kJ mol−1 using molecular dynamics simulations. The corresponding UV photon energy is sufficient to disrupt ˜10 lipid molecules to form a transient pore. Further, the disruption effect of motor actuations might be cumulative. Considering that the rotors (˜1 nm) are small relative to the thickness (7.5 to 10 nm) of the bilipid membranes, the rupture kinetics of the observed nanomechanical opening is not expected to be immediate. This is consistent with Applicants' experiments in this Example as well as the delayed membrane openings seen by others using probe-induced mechanical perturbations, accepting, however, that probe-tip perturbations are a vertical force model and hence considerably different than the tangential nanomechanical effects. Therefore, nanomechanical action can generate a concerted motion upon a 1-nm-long molecular rotor that will severely dislocate the membrane molecules, while other light absorbing molecules will merely dissipate the absorbed energy in random motions of atoms in the molecule, underscoring the efficacy of the nanomechanical effect for membrane disruption.
- The syntheses of the molecular machines were conducted as follows.
Molecular machines motor 11 and the correspondingalkynes - The synthesis of
motors machines - All glassware was oven-dried overnight prior to use. Reagent grade dichloromethane (CH2Cl2) was distilled from calcium hydride (CaH2) under N2 atmosphere. HPLC or spectroscopic grade water (H2O), chloroform (CHCl3) and acetonitrile (CH3CN) were used for the HPLC purification or the measurement of the optical properties. All reactions were carried out under N2 atmosphere unless otherwise noted. All other chemicals were purchased from commercial suppliers and used without further purification. The SNTRVAP-alkyne was prepared by BioPepTek (Malvern, Pa., USA). Flash column chromatography was performed using 230-400 mesh silica gel from EM Science. Thin layer chromatography (TLC) was performed using glass plates pre-coated with silica gel 40 F254 0.25 mm layer thickness purchased from EM Science. 1H NMR and 13C NMR spectra were recorded at 400, 500 or 600 and 100, 125 or 150 MHz, respectively. Chemical shifts (S) are reported in ppm from tetramethylsilane (TMS).
- An oven-dried 50 mL round-bottom flask equipped with a stir bar was charged with BODIPY-acid 17 (360.1 mg, 0.973 mmol), propargyl alcohol (0.23 ml, 3.89 mmol), EDC (279.8 mg, 1.46 mmol), DMAP (11.9 mg, 0.097 mmol) and CH2Cl2 (11 mL). The mixture was stirred at ambient temperature for 17 hours and concentrated under vacuum. The residue was purified by column chromatography (silica gel. CH2Cl2) to yield 13 as a red solid (272.7 mg, 69%): 1H NMR (600 MHz, CDCl3) δ 8.13 (dd, J1=7.9, J2=1.3 Hz, 1H), 7.69 (td, J1=7.5, J2=1.3 Hz, 1H), 7.59 (td, J1=7.7, J2=1.3 Hz, 1H), 7.36 (d, J=7.6 Hz, 1H), 5.95 (s, 2H), 4.71 (d, J=2.4 Hz, 2H), 2.55 (s, 6H), 2.39 (t, J=2.5 Hz, 1H), 1.32 (s, 6H). 13C NMR (150 MHz. CDCl3) δ 164.87, 155.11, 141.98, 140.94, 136.29, 133.30, 131.19, 131.10, 129.78, 129.66, 129.35, 121.08, 75.19, 52.80, 14.60, 14.06. HRMS (ESI) m/z calculated for [M+Na]+ C22H22N2O2BF2 429.1561, found 429.1556. The synthetic scheme is illustrated herein as
Scheme 2. - A 2 mL vial charged with motor diazide 11 (21.94 mg, 0.039 mmol), BODIPY dye 13 (35 mg, 0.086 mmol), CuSO4.5H2O(s) (0.97 mg, 0.0039 mmol) and sodium ascorbate (1.62 mg, 0.0117 mmol) was sealed with a rubber septum. A well-degassed mixture of CH2Cl2 (0.1 mL) and water (0.1 mL) was added to the vial, and the vial was shaken by a wrist-action shaking machine for 36 hours. The mixture was partitioned between CH2Cl2 (5 mL) and water (5 mL). The organic phase was dried over anhydrous MgSO4, filtered, and the filtrate was concentrated under vacuum. The crude product was purified by preparative TLC (silica gel, 4% MeOH in CH2Cl2) to afford the desired 2 as an orange solid (45 mg, 85%): 1H NMR (600 MHz, CD3CN) δ 8.00 (dd, J1=7.9, J2=1.1 Hz, 2H), 7.85-7.79 (m, 2H), 7.79-7.76 (m, 1H), 7.71 (s, 1H), 7.69-7.62 (m, 2H), 7.57-7.51 (m, 3H), 7.51-7.46 (m, 2H), 7.39-7.29 (m, 3H), 7.24-7.19 (m, 2H), 7.00 (dd, J1=8.1, J2=1.8 Hz, 1H), 6.84 (ddd, J1=8.2, J2=6.6, J3=1.3 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 6.59 (d, J=1.7 Hz, 1H), 6.02 (s, 1H), 6.01 (s, 2H), 5.99 (s, 1H), 5.27 (s, 2H), 5.14 (d, J=3.9 Hz, 2H), 4.58 (t, J=6.5 Hz, 2H), 4.36-4.30 (m, 1H), 4.30-4.24 (m, 1H), 4.20 (q, J=6.7 Hz, 1H), 3.68 (dd, J1=15.33, J2=6.11, 1H), 3.03 (t, J=6.5 Hz, 2H), 2.71 (t, J=6.5 Hz, 2H), 2.63 (d, J=15.5 Hz, 1H), 2.49 (s, 6H), 2.48 (s, 3H), 2.47 (s, 3H), 1.25 (s, 6H), 1.24 (s, 3H), 1.23 (s, 3H), 0.69 (d, J=6.9 Hz, 3H); 13C NMR (150 MHz, CD3CN) δ 165.77, 165.76, 155.38, 155.33, 148.01, 147.62, 143.11, 142.69, 142.66, 142.60, 142.16, 140.66, 138.08, 136.08, 136.00, 135.97, 134.72, 134.08, 134.06, 133.61, 131.62, 131.57, 131.28, 131.10, 131.01, 130.30, 130.27, 130.24, 130.17, 130.15, 129.83, 129.12, 128.71, 128.36, 128.33, 127.27, 125.88, 125.57, 124.94, 124.75, 124.72, 124.67, 122.11, 121.74, 121.60, 87.21, 86.41, 82.76, 82.31, 59.12, 58.99, 49.19, 48.98, 39.88, 38.47, 21.60, 21.31, 19.09, 14.35, 13.84, 13.82. HRMS (ESI) n/z calculated for [M+Na]+ C81H68N10O4B2F4S 1397.5184, found 1397.5210. The formed structure is illustrated herein as
Scheme 3. - DGEA-
alkyne peptide 14 was synthesized manually using standard solid-phase Fmoc protocols with Fmoc-amino acids and 4-pentynoic acid and was prepared as a C-terminal amide using Rink amide MBHA resin. Each acylation with Fmoc-amino acids or 4-pentynoic acid was performed using HATU (4 equiv) and N,N-diisopropylethylamine (DIPEA) (4 equiv) for 45 minutes in dimethylformamide (DMF) at room temperature, followed by Fmoc deprotection with 20% piperidine in DMF. DMF was used to wash the resin between each acylation and deprotection step. Cleavage from the resin was conducted by treatment with a mixture of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane (TIPS) at room temperature for 2 hours. The peptide was purified by reverse-phase HPLC and characterized by ESI-MS. HPLC Purification: Reverse-phase HPLC purification of 14 was performed on a Shimadzu CBM-20A instrument with Phenomenex Jupiter 4μ Proteo 90A (250×15 mm preparative) and Phenomenex Jupiter 4μ Proteo 90A (250×4.6 mm analytical) columns. The columns were eluted with a gradient of acetonitrile in water (10-60%) (flow rates of 8 mL/min and 1 mL/min for preparative and analytical columns, respectively). Trifluoroacetic acid (0.1%) was added to all eluents. The formed structure is illustrated herein asScheme 4. - A 1.5 mL Eppendorf tube charged with motor diazide 11 (2.0 mg, 0.0035 mmol), peptide-14 (2.46 mg, 0.0052 mmol), CuSO4.5H2O(s) (0.17 mg, 0.0007 mmol), sodium ascorbate (0.24 mg, 0.0017 mmol), and anhydrous DMF (60 μL) was bath sonicated for 10 hours at room temperature. After sonication, the crude product was purified by HPLC to afford 7 (2.5 mg, 47%) and 8 (2.0 mg, 53%) as white solids. HPLC Purification: Separation of 7 and 8 was performed using a reverse-phase peptide column (XBridge BEH300 prep C18, Part No. 186003628). Sample preparation involved dissolving the reaction mixture into 1 mL of a 1:1 mixture of H2O and CH3CN, followed by vigorous shaking and sonication. The mixture was then centrifuged at 14.000 rpm for 10 minutes to obtain a fully transparent supernatant for HPLC injection. This process was repeated on the pellet to extract as much product as possible, until the obtained supernatant resulted in no peaks in the HPLC chromatogram. A gradient elution system, containing 0.1% TFA in water and ACN, ramping from 20% to 90% of acetonitrile over 23 minutes with a flow rate of 2.5 mL/min at 40° C. was employed, which resulted in a baseline resolution of chromatogram peaks. The peaks were identified using a MicroToF ESI Mass Spectrometer. ESI m/z calculated for 7 [M]+ C73H80N16O18S 1502.6, found 1502.6. ESI m/z calculated for 7 [M]+ C54H53N11O9S 1032.4, found 1032.3. The structures of the peptides are illustrated herein as
Scheme 5. - A 1.5 mL Eppendorf tube charged with motor diazide 11 (1.88 mg, 0.00334 mmol), peptide-13 (4.0 mg, 0.00502 mmol), CuSO4.5H2O(s) (0.17 mg, 0.0007 mmol), sodium ascorbate (0.24 mg, 0.0017 mmol), and anhydrous DMF (70 μL) was sonicated for 10 hours at room temperature. After sonication, the crude product was purified by HPLC to afford 9 (2.4 mg, 43%) and 10 (2.0 mg, 57%) as white solids. HPLC Purification: Separation of 9 and 10 was performed using a reverse-phase peptide column (XBridge BEH300 prep C18, Part No. 186003628). Sample preparation involved dissolving the reaction mixture into 1 ml of 2:1 mixture of CH3CN and (CH3)2SO, followed by vigorous shaking and sonication. The mixture was then centrifuged at 14,000 rpm for 10 minutes to obtain a fully transparent supernatant for HPLC injection. This process was repeated on the pellet to extract as much product as possible, until the obtained supernatant resulted in no peaks in the HPLC chromatogram.
- A gradient elution system, containing 0.1% TFA in water and ACN, ramping from 20% to 100% of acetonitrile over 24 minutes with a flow rate 1.5 mL/min at 50° C. was employed, which resulted in a baseline resolution of chromatogram peaks. The peaks were identified using a MALDI Mass Spectrometer using CHCA matrix. MALDI m/z calculated for 9 [M+H]+ C101H133N24O24S 2153.97, found 2154.50. MALDI m/z calculated for 9 [M+H]+ C68H80N17O12S 1358.5893, found 1358.990. The structures are illustrated herein as
Scheme 6. - The monitoring of isomer interconversion of 8 is illustrated herein as
Scheme 7. - Premixed and dried synthetic phospholipid blend [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at a ratio of 5:3:2] were stored in a −20° C. freezer before use. To prepare the large unilamellar vesicles (LUVs), 5 mL 0.5× phosphate buffered saline (PBS) at pH 7.4 was added to swell the lipids solution (lipid concentration was 0.25 mg/mL). Then 7 μL of 210 μM BODIPY dye and 2 μL of 50 μM 1 (or 16 in the control experiments) were added to the solution. After incubation for 1 hour at room temperature with occasional vortexing, the lipids formed a cloudy suspension. The suspension was extruded more than 20 times with a mini-extruder (Avanti Polar Lipids) assembled with a polycarbonate membrane having a pore diameter of 600 nm. The resulting clear LUVs suspension was used immediately. The vesicles were then immobilized on a piece of cover-glass. Excessive BODIPY and 1 were washed off using fresh 0.5×PBS before the releasing experiments. The vesicles were then observed using a home-build confocal fluorescence microscope at 488 nm excitation and 535±50 nm emission.
- Due to the highly non-polar nature of
compound 1, it prefers to be sorbed in the bilipid membrane (FIG. 3 ).FIGS. 4A-D show that both BODIPY andcompound 1 were localized in the vesicle. The vesicles were then immobilized on a piece of cover-glass for the UV activation and releasing experiments. The UV light was provided by a UV LED at 365 nm with an intensity of ˜10 W·cm2 and analysis was done from 0 to 10-15 minutes. The fluorescence intensity of the BODIPY dye molecules in single vesicles was monitored using a confocal fluorescence microscope by continuously scanning a small area (20×20 μm2). - As the UV illumination time increased from 0 to 15 minutes, the fluorescence intensity of the vesicles became dimmer (
FIG. 4E ).FIG. 4F shows the normalized fluorescence intensity drop of 20 vesicles from 6 different sets of movies. Since the absolute intensity of the vesicles varied from vesicle to vesicle. Applicants normalized all the traces according to their initial intensities and analyzed the percent intensity drop. The error bar stands for the standard deviation of the normalized intensities. The first data point does not have an error bar. The average fluorescence intensity drop of 20 vesicles was 70±6% (mean±standard deviation) in the first 15 minutes of UV exposure. This is an observation based on many motor and dye molecules on individual vesicles. Even though the “gate” opening process is fast, the loss of dye molecules and their corresponding fluorescence is gradual and continuous, spanning a period as long as ˜15 minutes. - To exclude the possibility that the large fluorescence intensity drop was caused by (1) the UV light bleaching on BODIPY molecules, and (2) the thermal effect due to the absorption of UV light by 1. Applicants also performed a control experiment where 1 was replaced by 16 with the same concentration in the vesicles, 16 was used because it has a larger absorption coefficient at 365 nm than that of
compound 1. -
Compound 16 has two conjugated BODIPY molecules embedded in its molecular structure. However, its excitation and emission are both red-shifted by ˜70 nm as compared to those of isolated BODIPY molecules. Thus, its fluorescence is negligible to that from the isolated BODIPY molecules used as the probes when excited at 488 nm and collected at 535t 25 nm. -
FIGS. 4G-H show that compound 16 was localized in the vesicles. The fluorescence image ofcompound 16 was collected in the epi-fluorescence mode using 545±30 nm excitation and 605±55 nm emission. As a contrast, the fluorescence intensity drop of the BODIPY in 16-attached vesicles monitored in the confocal fluorescence mode (488 nm excitation and 535±25 nm emission) was much slower than in the 1-attached vesicles (FIGS. 4I-J ). The average fluorescence intensity drop from 20 selected vesicles (from 5 sets of movies) was 9%±20% (mean t standard deviation) for the first 15 minutes UV light exposure. Thus,compound 1 disturbs the lipid membrane upon UV-activation through a nanomechanical effect, which allows smaller BODIPY dye molecules to pass through the membrane. - A detailed investigation of the cellular behavior of each complex was conducted using PC-3, NIH 3T3, and CHO cell lines using fluorescence and laser scanning confocal microscopy. These cell lines were sourced from ATCC (NIH 3T3 CRL-1658, PC-3 CRL-1435 and CHO(-K1) CCL-61) and have been established and maintained in a
category 2 cell culture facility according to established standardized protocol for 12 months. The cells have been periodically monitored for mycoplasma contamination. - PC-3 has only been identified to cross contaminate other prostate adenocarcinoma cell lines but the source original PC-3 cell line has not been identified to be cross contaminated with any other cell line.
- Cells were maintained in exponential growth as monolayers in F-12/DMEM (Dulbecco's Modified Eagle Medium) 1:1 that was supplemented with 10% fetal bovine serum (FBS). Cells were grown in 75 cm2 plastic culture flasks, with no prior surface treatment. Cultures were incubated at 37° C. 10% average humidity and 5% (v/v) CO2. Cells were harvested by treatment with 0.25% (v/v) trypsin solution for 5 minutes at 37° C. Cell suspensions were pelleted by centrifugation at 1000 rpm for 3 minutes, and were re-suspended in fresh medium by repeated aspiration with a sterile plastic pipette.
- Microscopy cells were seeded in
untreated iBibi 100 μL live cell channels and allowed to grow to 40% to 60% confluence, at 37° C. in 5% CO2. At this stage, the medium was replaced and cells were treated with the studied nanomachines and co-stains as appropriate, with 0.1% DMSO (as detailed above) present in the final imaging medium. For live cell imaging. DMEM/F12 media (10% FBS) lacking phenol red was used from this point onwards. Following incubation, where Method B was used, the channels were washed with live cell imaging media and imaged using a purpose build incubator housing the microscope maintaining 37° C., 5% CO2 and 10% humidity. - All live cell imaging experiments used either NIH 3T3 mouse skin fibroblast cells, PC-3 cells, a
grade 3 human prostate adenocarcinoma, or Chinese hamster ovary cells (CHO). These are all well-studied cell lines with discrete morphological compositions. Each molecular machine was used as a stock solution at 0.10 to 1.00 μM with total dimethyl sulfoxide (DMSO) concentrations not exceeding 0.1% in the final cell media in order to avoid unwanted cell membrane permeabilization, the DMSO being required for solubility of the organic nanomachines. All loading experiments are carried out in a light-suppressed manner and the possibility of induced or accelerated uptake due to interaction between the molecular motors and the applied co-stain has been eliminated using a series of individual and reversed loading experiments. - Cell toxicity was determined using a ChemoMetec A/S NucleoCounter 3000-Flexicyte instrument with Via1-cassette cell viability cartridge using the cell stain Acridine Orange for cell detection, and the nucleic acid stain DAPI for detecting non-viable cells and Annexin V for the detection of apoptosis.
- In cellular uptake studies, cells were seeded in 6-well plates and allowed to grow to 80% to 100% confluence at 37° C. in 5% CO2. Culture medium was then replaced with culture medium containing 0.1% DMSO with individual nanomachines 1-10 for 24 hours at 0.10, 0.50 and 1.00 mM.
- All cell colonies bearing nanomachines displayed 92±5% viability. The control blank cells were established at 95±3% viability. In addition to 0.1% DMSO being used for molecular machine introduction, all washing solutions also contained 0.1% DMSO. At this concentration. DMSO does not affect the cells. This was determined by control experiments using all imaged cell lines cultured in DMSO-free and 0.1% DMSO-containing cell media while establishing the initial control UV-induced cell death parameters. To confirm the non-activated low toxicity of these molecular machines, all live cell imaging samples with all three studied cell lines were re-incubated, using Method A, in the dark and re-imaged using only transmission microscopy using a tungsten bulb with a LP 420 nm cut off filter. These experiments confirmed that all previously non-UV-exposed cells, regardless of the cell line studied, still proliferated in the presence of the molecular motor stock solutions for up to 72 hours using visible light at a pre-set time point to assess cell morphological changes along with viability and vitality.
- All experimental imaging parameters (i.e., laser beam size, confocal pinhole size, laser intensity, line scanning speed, scanning area (field of view. FOV), and line averaging sequences) were kept constant throughout the experiments. The accuracy and errors associated with the establishment of accelerated necrosis are determined based on one dual channel imaging sequence, which takes 15 seconds in total. This imaging sequence has been carefully established using untreated live cells.
- The optimized imaging parameters allow appropriately high scanning speed to follow natural homeostatic events and identify any induced morphological or fluorescent signal localization change. Meanwhile, they also allow sufficiently long integration time for each pixel so an adequate amount of photon signals can be collected. In order to satisfy the Nyquist sampling criteria, the pixel size is set as ⅕ of the laser spot size. The images were acquired using a bidirectional 2-line averaging sequence, which gives minimal dead time (<1 ms) between line scanning. The image size was adjusted to 100×100 μm in order to study 1 to 3 cells simultaneously. Each individual experiment was repeated 3 times on triplicate slides. On each slide, at least 5 well-separated areas were imaged (Table 2).
- Steady state fluorescence images were recorded using a PhMoNa enhanced Leica SP5 II LSCM equipped with a HCX PL APO 63×/1.40 NA LambdaBlue Oil immersion objective. As an initial control experiment to establish the UV-induced cell toxicity threshold, while mitigating voxel exposure and ensuring sufficient laser dwell time, an imaging sequence has been established to monitor cell morphological and physiological changes as a function of time. All experiments used cell-line-determined culture medium containing 0.1% DMSO.
- The applied 100 Hz detection sequence was based on a bidirectional dual-channel continuous scanning method where a minimalistic non-damaging visible laser light (458 nm, 0.2 mW) is used in conjunction with the above detailed UV exposure. This is set as a 2 line/scan accumulation parallel acquisition sequence that is recorded as a function of time. Studied channels correspond to transmission images and UV-induced mitochondrial auto-fluorescence detected at 460 to 550 nm. Applicants further confirmed that the effectiveness of monitoring the UV-activated nanomechanical action on live cells using a conventional CW mercury-arc excitation source equipped with an epi-fluorescence setup consisting of a Zeiss Axiovert 200M inverted microscope, as discussed in
FIGS. 18A-C . - The modular PhMoNa technique is based on a laser scanning confocal microscope (LSCM) harnessing spatially modulated illumination intensities, using an in situ generated raster-scanned standing wave excitation beam optical grid pattern. Such an approach allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2 (x,y=62 nm, z=188 nm @ 355 nm Ex 63×1.40NA, 100 Hz/line, 200 nJ/voxel dwell time) and is free from time-consuming post-image processing deconvolution algorithms.
- Live cell experiments have been performed on the above detailed custom built PhMoNa system based on this Leica SP5 II platform operating with a fiber-coupled 355 nm Coherent laser (Nd:
YAG 3rd Harmonic, 80 mW) for UV activation of the motor. Steady state fluorescence images were recorded using the PhMoNa enhanced Leica SP5 II LSCM confocal microscope equipped with a HCX PL APO 63×/1.40 NA LambdaBlue Oil immersion objective. - Data were collected using 2× digital magnification at 100 Hz/line scan speed (4-line average, bidirectional scanning) at 355 nm (3 harmonic NdYAG laser, set at 20 mW, 400 nJ/voxel total dwell time). In order to achieve excitation with maximal probe emission, the microscope was equipped with a triple channel imaging detector, comprising a conventional PMT systems and two HyD hybrid avalanche photodiode detectors. The latter parts of the detection system, when operated in the BrightRed mode, is capable of improving imaging sensitivity by 25%, reducing signal to noise by a factor of 5. Frame size was determined at 1024×1024 pixel, with ×2 digital magnification to ensure illumination flatness of field and 0.6 airy disc unit determining the applied pinhole diameter rendering on voxel to be corresponding to 62×62 nm2 (frame size 125×125 μm2) with a section thickness set at 188 nm (at 355 nm excitation). A HeNe or Ar ion laser was used to aid transmission image capture and when commercially available organelle-specific stains (e.g. MitotrackerRed™ or PI) were used to corroborate cellular compartmentalization or follow the onset of necrosis
- All imaging parameters were kept constant across experiments. This includes voxel size, laser power, line speed and averaging sequences, unless otherwise noted. The accuracy and errors associated with the establishment of accelerated necrosis is one frame dual channel imaging sequence that totals 15 seconds.
- The threshold algorithm to control brightness automated by the Leica SP5 II software is calculated by the image specific signal-to-noise ratio or it is accessible post image-processing.
- Although PI's molar extinction coefficient is relatively low compared to other organic cell dyes, upon intercalation, its red fluorescence emission intensity increases by more than 10 times, thereby becoming a suitable counter-stain to assess and establish the onset of UV-induced necrosis. In light of its favorable spectroscopic properties, no major alteration of the experimental parameters are needed other than exchange of the 488 nm laser line, used to generate transmission images, to 543 nm (0.2 mW, also confirmed to be non-disruptive to cells).
- When PC-3 cells are loaded at 0.5 or 1.0 μM with 9 or 10, washed three times (Method B) or unwashed (Method A) after 24 hours of incubation, the cells visually appear without major signs of toxicity. However, the cells which had been incubated with 9 or 10 over 24 hours were not dividing as prolifically. The cultures appear to be populated ˜25% less in the presence of 9 and 20% of the cells in 10 compared to control untreated cells (
FIGS. 12A-E ). - Upon subsequent transmission microscopy verification, this was found to be in agreement with the amount of mobilized cells present with the experiments in the cell culture media. In order to further investigate this point, these cells were collected via gentle centrifugation and re-suspended in fresh PBS and analyzed by Image Cytometry. Using Annexin V, these cells were all confirmed to show signs of apoptosis at 93±5%. This is in contrast to the remaining coverslip surface-bound cells that appear healthy.
- After harvesting the cells via trypsination (0.25% v/v), subsequent tests showed no sign of cell apoptosis (<5%). These latter types of non-apoptotic cells show identical percentage of necrosis acceleration upon subsequent UV-activation of the cell-surface bound nanomachines compared to their shorter incubation time analogues. Thus, it appears that, upon 24 hour incubation, a noticeable amount of cells have nanomachines 9 and 10 either bound to vital cell membrane channels or transport proteins or they are internalized, subsequently shutting down some natural homeostatic mechanisms that leads to programmed cell death (
FIGS. 12A-E ). Furthermore, this is not the case with shorter incubation times regardless of whether Method A or B is used. - In a separate experiment, PC-3 cells are also incubated for 30 minutes to 4 hours with 9 or 10, and then washed three times and further incubated for 24 hours in rotor free media to see if they retain their surface-bound molecular rotors. The cells are then UV-activated to see if necrosis would be induced at the previously established accelerated time points.
- However, UV-activated nanomechanical accelerated necrosis did not occur to the same extent as seen before. Some cells lose their bound motor activity or the motors themselves were lost. The cells die only 25% faster rather than the expected 40 to 50% accelerated necrosis since there is no constant 0.5 or 1.0 μM nanomachine exposure of the cells in the culture media (i.e. constant flux). In these experiments, cell proliferation is comparable (within 5%) to the control molecular motor-free cells.
- In summary, the surface bound rotors upon 24 hour incubation only induce a noticeable amount of apoptotic cell toxicity via suspected internalization or membrane adhesion if there is a constant flux of nanomachines in the medium. Nanomachines 9 and 10 appear to detach from the cell-surface with their lack of concentration gradient from the media. Hence, there is no triggered apoptosis. There is no difference between 9 and 10 in these experiments (
FIGS. 12A-13E ). - The control molecular motor-free NIH 3T3 cells and the cells loaded with either 9 or 10 (0.5 to 1.0 μM) are identical without any change in behavior or onset of UV-induced nanomechanical necrosis. This is the same as in overnight media incubation. The cells show signs of proliferation of 10 to 25%. NIH 3T3 cells incubated for 9 for 24 hours seem to be the same as the cells exposed for 1-2 hours with no change in rates upon UV-accelerated nanomechanical necrosis compared to motor-free blanks (
FIGS. 13A-E ). - Thus, 9 does not diffuse into NIH 3T3 cells and thereby have nanomechanical-induced necrosis or apoptosis. Cell proliferation is slower than blank motor-free cells but that could be the result of 9 binding to essential proteins or supplements in the media, slowing natural cell homeostasis. NIH 3T3 cells incubated with 10 for 24 hours are similar to those incubated with 9 except that in the case of 10, the NIH 3T3 cells did not proliferate as much and upon UV irradiation. Rather, the cells die 15 to 20% faster with necrosis than untreated motor-free blanks (
FIGS. 14A-E ). Hence, mono-peptide-bearingnanomachine 10 can be internalized or membrane bound upon 24 hours of incubation, but in smaller quantities that do not trigger any noticeable toxic effect that could lead to programmed apoptotic cell death, while 10 only accelerates necrosis with less than half the efficiency compared to identical experiments using the targeted PC-3 cells. - In order to further confirm the selectivity of peptide-bearing
rotor compounds nanomachines FIGS. 15A-E ). - An evaluation of the forces produced by the UV-activated molecular machines upon the bilipid membranes is considered here. Membrane rupture and pore formation under a tangential mechanical force have been studied theoretically and experimentally. Generally, when the stress of the membrane exceeds their critical value, membrane rupture occurs. The critical rupture tension of biological membranes varies from 1 to 30 mN m−1, depending on the specific chemical composition of the membrane. It has also been shown that the rupture stress is dependent on the stress loading rates. For example, it has been shown that in impulse stretching experiments over tens of μs, red blood cell membranes can sustain the stress by ten times over stresses induced by quasi-static stretching conditions. Further, pore formation is a highly dynamic process whereupon the pores either close or continue to grow until rupture of the membrane. Under tension, pore formation becomes faster, though the pore formation is a transient event, which is challenging to capture.
- Based upon the studies performed, the following features appear preferred for molecular machine opening of cellular membranes. A rapidly spinning rotor is preferred because no membrane perturbation was observed without rotation. All of the nanomachines studied here have rotors that can rotate in a 2 to 3 MHz regime, and there was sufficient rotary actuation for disruption.
- Moreover, smaller and addend-unencumbered molecular machines are preferred over the more encumbered systems. The molecular machines preferably embed in the membranes to show opening because their mere presence in the medium may not be sufficient in all circumstances.
- When targeting, it is preferred to have a targeting addend that does not impede rotor operation. If using two relatively large addends, they might retard the rotor from interacting with the lipid bilayer, thereby slowing the nanomechanical perturbation of the membrane. Since these are molecular-sized, pore formation on the membranes is not immediate. The process can take about 1 minute to become detectable through leakage currents and twice that long based upon morphological changes. Sufficient rupture stress will have to be displayed by the rotors in order to be effective in bilayer disruption. Finally, shorter UV-actuation times of <30 scan permit analytes in the medium to enter the cells before the cells can reach the stage of programmed cell death.
- Although all of the nanomachines studied here have rotors that can rotate in the 2 to 3 MHz regime. Applicants did not always excite the motors to their full speeds. In the confocal imaging experiments, the UV illuminations were ˜10 MW cm−1, exceeding the motor saturation illumination level, which is ˜10 kW cm−2, assuming that the motor absorption cross section is 10 16 cm2. In the synthetic vesicle releasing experiments and patch clamp studies. Applicants were using only ˜10 W cm−2 UV illumination power. On the other hand, the UV illumination was continuous in the synthetic vesicle and patch clamp studies while the confocal illumination was intermittent.
- Therefore, the total dosage of UV illumination was on a similar level across the different experiments in this study. Accordingly, it is likely that the total dosage of UV illumination is an important parameter since Applicants observed the membrane rupture on similar time scales across the three experiments: synthetic vesicles, confocal imaging on three different live cells types, and whole patch clamp on a fourth live cell type.
- In summary, nanomechanical action can disrupt external or internal cellular membranes and it can be used to introduce analytes into cells or expedite cell death. By synthetic design, the nanomachines can be tracked within a cell or used to target specific cells through unique cell-surface recognition elements. The efficacy of this method for in vitro studies was demonstrated. Extensions to in vivo applications can be envisioned, especially at locations where short UV-exposure is acceptable (e.g., dentistry, localized epidermis and colorectal treatments). The use of molecular motors that are activatable by two-photon-, near-infrared- or radio-frequency-inputs, would make broader in vivo treatments viable.
- In this Example, Applicants demonstrate that a two-photon illumination in the near-infra red (IR) region can activate
molecular motors FIGS. 2C, 2D-1 , and 2D-2), thereby resulting in Propidium Iodide (PI) dyes entering NIH 3T3 orPC 3 cells. Two-photon microscopy studies of NIH 3T3 cells were executed on a Nikon E600 upright confocal microscope coupled to a tuneable (710-950 nm Coherent MaiTai) multiphoton source operating at (20%) 240 mW power using a 166 lps scan speed and 128×128 pixel frame resolution for MP-illumination. Images (512×512 pixel, 166 lps) were recorded using a 543 nm (1 mW HeNe) laser combined with a 560 nm long-pass filter to record PI (200 nM) fluorescence and follow the onset of induced necrosis. - The results are shown in
FIGS. 19A-D . In particular.FIG. 19A shows images of cells in live cell media (10% FBS) with no molecular motors present.FIG. 19B shows images of identical cells but with a 1 μM control molecular motor 5 (structure shown inFIG. 2C and described in Example 1 as lacking a rotor but still absorbing UV-light) using Method A (10 minute pre-incubation prior to imaging, as described in Example 1).FIG. 19C shows images of identical cells but with 1 μM ofmolecular motor 3 present using Method A (10 minute pre-incubation prior to imaging, as described in Example 1).FIG. 19D shows images of PC-3 cells with 1.0 μM ofmolecular motor 8 present using Method B (1 hour pre-incubation followed by washing and subsequent imaging, as described in Example 1). - Two photon live cell microscopy studies were also executed on a Nikon E600 upright confocal microscope coupled to a tuneable (710-950 nm Coherent MaiTai) multiphoton source operating at (19.8%) 240 mW power at 720 nm using 166 lines/sec scan speed and 128×128 pixel frame resolution for MP-illumination. An oil immersion Nikon IR objective (×60/1.4NA) was used. Images (512×512 pixel, 166 l/s) were recorded using a 543 nm (1 mW HeNe) laser combined with a 570 nm long-pass filter to record PI dyes (200 nM). Fluorescence was measured as 2 frame averaged images in order to follow the onset of induced necrosis. Necrosis times have been assigned to detectable PI signals in the nucleolus.
- The results are shown in
FIGS. 20A-L , where the scale bars correspond to 20 μm. Time points correspond to duration of continuous multi-photon exposure in 1 minute segments only paused for PI imaging.FIG. 20A shows images of control NIH 3T3 cells in live cell media (10% FBS) with no molecular motors present.FIG. 20B shows images of identical NIH 3T3 cells with 1 μM ofmolecular motor 5 present using Method A.FIG. 20C shows images of identical NIH 3T3 cells with 1 μM ofmolecular motor 3 present using Method A.FIG. 20D shows images of NIH 3T3 cells with 1 μM ofmolecular motor 8 present using Method B (1 hour pre-incubation prior washing and subsequent imaging).FIG. 20E shows images of identical control PC3 cells with no molecular motors present.FIG. 20F shows images of identical PC3 cells with 1 μM ofmolecular motor 3 present using Method A.FIG. 20G shows images of identical PC3 cells with 1 μM ofmolecular motor 5 present using Method A.FIG. 20H shows images of identical PC3 cells with 1 μM ofmolecular motor 3 present using Method B.FIG. 20I shows images of identical PC3 cells with 1 μM ofmolecular motor 8 present using Method A.FIG. 20J shows images of identical PC3 cells with 1 μM ofmolecular motor 9 present using Method A.FIG. 20K shows images of identical PC3 cells with 1 μM ofmolecular motor 8 present using Method B.FIG. 20L shows images of identical PC3 cells with 1 μM ofmolecular motor 9 present using Method B. - Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.
Claims (10)
1. A molecule having the following structure:
wherein R1 and R2 are each independently selected from the group consisting of hydrogen, alkanes, alkenes, alkynes, carboxyl groups, ketone groups, alkoxy groups, methoxy groups, ethers, nitro groups, nitriles, sulfates, sulfonates, halogens, amine groups, amide groups, alcohols, aromatic groups, aryl groups, phenyl groups, annulated rings, carbohydrates, polysaccharides, peptides, targeting agents, tracing agents, fluorophores, solubilizing agents, active agents, and combinations thereof;
wherein X is selected from the group consisting of S, CH2, and O,
wherein R3 has one of the following structures:
2. The molecule of claim 1 , having the following structure:
wherein R1 and R2 are each independently selected from the group consisting of hydrogen, alkanes, alkenes, alkynes, carboxyl groups, ketone groups, alkoxy groups, methoxy groups, ethers, nitro groups, nitriles, sulfates, sulfonates, halogens, amine groups, amide groups, alcohols, aromatic groups, aryl groups, phenyl groups, annulated rings, carbohydrates, polysaccharides, peptides, targeting agents, tracing agents, fluorophores, solubilizing agents, active agents, and combinations thereof.
3. The molecule of claim 1 , wherein R1 and R2 are each independently selected from the group consisting of hydrogen, alkanes, alkenes, alkynes, nitro groups, nitriles, amine groups, amide groups, annulated rings, carbohydrates, polysaccharides, peptides, targeting agents, tracing agents, fluorophores, solubilizing agents, active agents, and combinations thereof, and X is S.
4. The molecule of claim 3 , wherein R1 and R2 are each independently selected from the group consisting of hydrogen, alkanes, alkenes, alkynes, nitro groups, nitriles, amine groups, amide groups and combinations thereof.
7. The molecule of claim 1 , comprising a targeting agent for directing the molecule to a desired lipid bilayer, optimally wherein the targeting agent is selected from the group consisting of amino acids, peptides, proteins, aptamers, antibodies, small molecules, carbohydrates, polysaccharides, and combinations thereof.
8. The molecule of claim 1 comprising a tracing agent for tracking the molecule, optionally wherein the tracing agent is selected from the group consisting of fluorophores, chromophores, dyes, radio-labeled molecules, radioactive nuclei, high contrast agents, gadolinium, gallium, thallium, fluorinated compounds, and combinations thereof.
9. The molecule of claim 1 comprising a solubilizing agent for maintaining the water solubility of the molecule, optionally wherein the solubilizing agent is selected from the group consisting of peptides, glycols, alcohols, carboxylates, polysaccharides, salts, acids, polyethers, polyethylene glycols (PEGs), carbohydrates, and combinations thereof.
10. The molecule of claim 1 comprising an active agent, optionally wherein the active agent is releasably associated with the molecule, and/or optionally wherein the active agent is selected from the group consisting of drugs, peptides, polypeptides, nucleotides, DNA, RNA, siRNA, enzymes, and combinations thereof.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18/149,934 US20230149567A1 (en) | 2016-07-14 | 2023-01-04 | Mechanical opening of lipid bilayers by molecular nanomachines |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662362206P | 2016-07-14 | 2016-07-14 | |
PCT/US2017/042148 WO2018013930A1 (en) | 2016-07-14 | 2017-07-14 | Mechanical opening of lipid bilayers by molecular nanomachines |
US201916316716A | 2019-01-10 | 2019-01-10 | |
US17/233,102 US11565003B2 (en) | 2016-07-14 | 2021-04-16 | Mechanical opening of lipid bilayers by molecular nanomachines |
US18/149,934 US20230149567A1 (en) | 2016-07-14 | 2023-01-04 | Mechanical opening of lipid bilayers by molecular nanomachines |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/233,102 Continuation US11565003B2 (en) | 2016-07-14 | 2021-04-16 | Mechanical opening of lipid bilayers by molecular nanomachines |
Publications (1)
Publication Number | Publication Date |
---|---|
US20230149567A1 true US20230149567A1 (en) | 2023-05-18 |
Family
ID=60953363
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/316,716 Active 2037-12-19 US11154623B2 (en) | 2016-07-14 | 2017-07-14 | Mechanical opening of lipid bilayers by molecular nanomachines |
US17/233,102 Active US11565003B2 (en) | 2016-07-14 | 2021-04-16 | Mechanical opening of lipid bilayers by molecular nanomachines |
US18/149,934 Pending US20230149567A1 (en) | 2016-07-14 | 2023-01-04 | Mechanical opening of lipid bilayers by molecular nanomachines |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/316,716 Active 2037-12-19 US11154623B2 (en) | 2016-07-14 | 2017-07-14 | Mechanical opening of lipid bilayers by molecular nanomachines |
US17/233,102 Active US11565003B2 (en) | 2016-07-14 | 2021-04-16 | Mechanical opening of lipid bilayers by molecular nanomachines |
Country Status (4)
Country | Link |
---|---|
US (3) | US11154623B2 (en) |
EP (1) | EP3484529A4 (en) |
CA (1) | CA3034234A1 (en) |
WO (1) | WO2018013930A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11154623B2 (en) | 2016-07-14 | 2021-10-26 | William Marsh Rice University | Mechanical opening of lipid bilayers by molecular nanomachines |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7964722B2 (en) * | 2002-08-31 | 2011-06-21 | Board Of Regents Of The Nevada System Of Higher Education, On Behalf Of The University Of Nevada, Reno | Light-driven rotary molecular motors |
JP5133034B2 (en) * | 2007-05-24 | 2013-01-30 | 日東電工株式会社 | Optical film, image display device, diethynylfluorene and polymer thereof |
US10363309B2 (en) * | 2011-02-04 | 2019-07-30 | Case Western Reserve University | Targeted nanoparticle conjugates |
EP2489371A1 (en) * | 2011-02-18 | 2012-08-22 | Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria | Carrier peptides for drug delivery |
US10426850B2 (en) * | 2011-11-22 | 2019-10-01 | The Johns Hopkins University | Collagen mimetic peptides for targeting collagen strands for in vitro and in vivo imaging and therapeutic use |
CN108042811A (en) | 2012-11-15 | 2018-05-18 | 恩多塞特公司 | For treating the conjugate of the disease as caused by PSMA expression cells |
US11154623B2 (en) | 2016-07-14 | 2021-10-26 | William Marsh Rice University | Mechanical opening of lipid bilayers by molecular nanomachines |
-
2017
- 2017-07-14 US US16/316,716 patent/US11154623B2/en active Active
- 2017-07-14 CA CA3034234A patent/CA3034234A1/en active Pending
- 2017-07-14 WO PCT/US2017/042148 patent/WO2018013930A1/en unknown
- 2017-07-14 EP EP17828535.9A patent/EP3484529A4/en active Pending
-
2021
- 2021-04-16 US US17/233,102 patent/US11565003B2/en active Active
-
2023
- 2023-01-04 US US18/149,934 patent/US20230149567A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
EP3484529A4 (en) | 2020-04-08 |
US11565003B2 (en) | 2023-01-31 |
US20190290785A1 (en) | 2019-09-26 |
CA3034234A1 (en) | 2018-01-18 |
US20210252166A1 (en) | 2021-08-19 |
WO2018013930A1 (en) | 2018-01-18 |
US11154623B2 (en) | 2021-10-26 |
EP3484529A1 (en) | 2019-05-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhang et al. | Imaging lysosomal highly reactive oxygen species and lighting up cancer cells and tumors enabled by a Si-rhodamine-based near-infrared fluorescent probe | |
Yang et al. | Combating bacterial infection by in situ self-assembly of AIEgen-peptide conjugate | |
Kim et al. | Two-photon absorbing dyes with minimal autofluorescence in tissue imaging: application to in vivo imaging of amyloid-β plaques with a negligible background signal | |
Hu et al. | Noncanonical amino acids for hypoxia-responsive peptide self-assembly and fluorescence | |
Zhang et al. | Mechanistic study of IR-780 dye as a potential tumor targeting and drug delivery agent | |
Okuda et al. | 2-Nitroimidazole-tricarbocyanine conjugate as a near-infrared fluorescent probe for in vivo imaging of tumor hypoxia | |
Zhuang et al. | Esterase-activated theranostic prodrug for dual organelles-targeted imaging and synergetic chemo-photodynamic cancer therapy | |
Jiao et al. | Cyclodextrin-based peptide self-assemblies (Spds) that enhance peptide-based fluorescence imaging and antimicrobial efficacy | |
Gregersen et al. | Intracellular delivery of bioactive molecules using light-addressable nanocapsules | |
Zhang et al. | In vivo and in situ activated aggregation-induced emission probes for sensitive tumor imaging using tetraphenylethene-functionalized trimethincyanines-encapsulated liposomes | |
Cao et al. | Synthesis and evaluation of a stable bacteriochlorophyll-analog and its incorporation into high-density lipoprotein nanoparticles for tumor imaging | |
Zhang et al. | Amino-Si-rhodamines: a new class of two-photon fluorescent dyes with intrinsic targeting ability for lysosomes | |
Saha et al. | Targeting and imaging of mitochondria using near-infrared cyanine dye and its application to multicolor imaging | |
US20230149567A1 (en) | Mechanical opening of lipid bilayers by molecular nanomachines | |
CN103502465A (en) | Method for detecting cancer cell using fluorescently labeled L-glucose derivative, and cancer cell-imaging agent comprising fluorescently labeled L-glucose derivative | |
An et al. | Human glioblastoma visualization: triple receptor-targeting fluorescent complex of dye, SIWV tetra-peptide, and serum albumin protein | |
Liu et al. | Biomineralization of aggregation-induced emission-active photosensitizers for pH-mediated tumor imaging and photodynamic therapy | |
Firsov et al. | Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor | |
Lee et al. | A bright blue fluorescent dextran for two-photon in vivo imaging of blood vessels | |
Mao et al. | Spectroscopic techniques for monitoring stem cell and organoid proliferation in 3D environments for therapeutic development | |
Li et al. | Functional molecules and nano-materials for the Golgi apparatus-targeted imaging and therapy | |
Gilson et al. | Trafficking of a single photosensitizing molecule to different intracellular organelles demonstrates effective hydroxyl radical-mediated photodynamic therapy in the endoplasmic reticulum | |
Liu et al. | Highly efficient cell membrane tracker based on a solvatochromic dye with near-infrared emission | |
Xu et al. | Lipid droplet formation and dynamics: tracking by time-resolved fluorescence imaging | |
US20200340920A1 (en) | Hydrophilic silicon-rhodamine fluorescent probes and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |