Unprecedented inhibition of tubulin polymerization directed by gold nanoparticles inducing cell cycle arrest and apoptosis

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1 Eletroni supplementry informtion (ESI) for Nnosle Unpreedented inhiition of tuulin polymeriztion direted y gold nnoprtiles induing ell yle rrest nd poptosis Diptimn Choudhury,,e, Pulrjpilli Lourdu Xvier,, Kmlesh Chudhri, d Roin John, Anjn Kumr Dsgupt, Thlppil Prdeep * nd Gopl Chkrrti * Deprtment of Biotehnology nd Dr. B. C. Guh Centre for Geneti Engineering nd Biotehnology, University of Clutt, 35 Bllygunge Cirulr Rod, Kolkt, West Bengl, Indi 719. *Emil: gg@luniv.in Deprtment of Biohemistry, University of Clutt, 35 Bllygunge Cirulr Rod, Kolkt, West Bengl, Indi 719. Deprtment of Chemistry nd DST Unit of Nnosiene, Indin Institute of Tehnology Mdrs, Chenni, Tmil Ndu, Indi 636. *Emil: prdeep@iitm..in d Deprtment of Biotehnology, Indin Institute of Tehnology Mdrs, Chenni, Tmil Ndu, Indi 636. e Currently t Deprtment of Mediine, Division of Uro-Onology, Cedrs Sini Medil Centre, 87-Bevrly Blvd, Los Angeles, Cliforni 948, USA. these uthors ontriuted eqully to this work 1

2 Eletroni Supplementry Informtion 1 λ A mx =537 nm λ A mx =532 nm λ A mx =525 nm 6 nm 5 nm 4 nm 2 nm 5 nm 5 nm Fig. S1 Extintion spetr of three different sized (2 nm, 4 nm nd 6 nm) itrte pped AuNPs with orresponding TEM imges, nmely AuNP 2, AuNP 4 nd AuNP 6. Sle r for ll TEM imges is 5 nm. 2

3 Eletroni Supplementry Informtion 1A: Clultion of molrity of AuNP Molrity of AuNPs in solution ws lulted using the following formul, ( ) ( ) ( ) = ( 6 * 25 * 197 * 7 ) / ( 22 * (4 * 1-7 ) 3 * 19.3 * 6.23 * 1 23 ) = 2685 / ( 22 * 64 * 1-21 * 19.3 * 6.23 * 1 23 ) = 2685 / ( 22 * 64 * 19.3 * 6.23 * 1 5 ) = 2685 / ( * 1 5 ) = *1-5 µm = pm whih gives molrity of AuNP 4 in the stok solution s pm. Where, M = Molrity of Au 3+ stok in µm A r = Atomi weight of Au in g D = Dimeter of nnoprtile in m ρ = Density of gold in g/m 3 N A = Avogdro numer For AuNP 4 molrity of stok solution ws found to e, pm. 3

4 Optil Rottion (mdeg) % of polymeriztion Eletroni Supplementry Informtion 2 The extents of polymeriztion inhiition were round 28.6 ± 2.7%, 4.32 ± 1.7% nd 6.47 ± 3.5% in presene of AuNP 4 t 5, 12.5 nd 25 pm, respetively (for 3 minutes). Hene the lulted IC 5 vlue (i.e. 5% inhiitory onentrtion) for AuNP 4 ws 18.6 ±.9 pm IC 5 = pm Conentrtion of AuNP 4 (pm) Fig. S2 Plot showing the lultion of IC 5 onentrtion of AuNP 4 for purified mmmlin tuulin polymeriztion. Error in the determintion of % polymeriztion is given in supporting informtion 1. From the ove plot (Fig. S2) we n infer tht t the IC 5 dose of AuNP 4 for polymeriztion inhiition of tuulin (12 µm) is ~18.6 pm nd the molr rtio of AuNP 4 : tuulin is round 1 : 3.16 X 1 5. Eletroni Supplementry Informtion pm AuNP pm AuNP 4 25 pm AuNP Wvelength (nm) Fig. S3 Cirulr dihroism spetrosopi mesurements reveling no signifint hnge in the seondry struture of tuulin upon interting with AuNP 4. 4

5 Normlized Trnsmittne Normlized Trsmittne Eletroni Supplementry Informtion 4 A Mirotuule AuNP-Mirotuule Wvenumer (m -1 ) B Tuulin AuNP 4 -Tuulin Wvenumer (m -1 ) Fig. S4 FTIR spetr of mirotuule nd tuulin (solid lk line in A nd B) nd AuNP 4 treted tuulin nd mirotuule (red solid line in A nd B). 5

6 412 nm Fluoresene Intensity (.u.) Intensity t 335 nm Eletroni Supplementry Informtion 5 25k 2k 15k k 5k min 1 min 2 min 5 min 1 min 2 min 3 min 4 min 5 min Wvelength (nm) Time (minutes) Fig. S5 PL spetr showing the quenhing of intrinsi fluoresene of tryptophn upon intertion with AuNP 4. Eletroni Supplementry Informtion pm 12.5 pm 25 pm Time (s) Fig. S7 Thiol estimtion with ontrol nd tuulin inuted with AuNP 4 t different onentrtions. Results indited 3-5% loss of ysteine ontent per heterodimer. 6

7 Eletroni Supplementry Informtion 7 Cell Viility (%) Sttering t 35 nm MT+Buffer Without orretion % of Polymeriztion A.3.2 MT+Buffer MT+5pM AuNP MT pm AuNP MT +25 pm AuNP After orretion 25 pm AuNP 4 B MT+5 pm AuNP 4 MT+12.5 pm AuNP 4 MT+ 25 pm AuNP 4.1. Sttering from AuNPs dded fter 25 mins 12.5 pm AuNP 4 5 pm AuNP 4 Buffer Time (min) 2 Fig. S7 A) UV-vis spetrosopi study of effet of Au NPs on the polymerized tuulin. After 25 minutes of polymeriztion, upon ddition Au NPs did not depolymerise the polymerized mirotuules in vitro, s oserved from the spetr (highlighted spetrl region with dotted ellipse; the originl spetr were sutrted with the orresponding sttering spetr of Au NPs for lrity). B) Brdigrm showing the perentge of retention of polymerized tuulins with vrious onentrtions of Au NP. Eletroni Supplementry Informtion 8 A549 ells Clulted IC 5 = pm 1 2 AuNP 4 (pm) Fig. S8. Cell viility ssy results of A549 ells upon 72 h AuNP 4 tretment. The lulted IC 5 vlue ws 29.5±1.7 pm. 7

8 Sttering intensity (.u.) Eletroni Supplementry Informtion 9A e µm µm d Wvelength (nm) 1 µm 2 µm Fig. S9A. Drk field mirosopi (DFM) imges of AuNP 4. Left side: imges, nd re DFM imges of AuNP 4 nd d is the lrge re imge from whih, nd were seleted. Right side (e): the orresponding Plsmon Resonne Rleigh Sttering (PRRS) spetr of nnoprtiles: (lue solid line representing AuNP in the imge ), (lk solid line representing AuNP in the imge ), nd (red solid line representing AuNP in the imge ). These prtiles re lelled in imges, nd. 8

9 Sttering intensity (.u.) Eletroni Supplementry Informtion 9B d 3 1 µm 2 1 µm Wvelength (nm) 1 µm Fig. S9B. AuNPs in the ggregted tuulin mtrix. Left: imges, nd re seleted re from Figure 2D. is prt of tuulin ggregte without nnoprtile, nd re nnoprtiles in the ggregted protein mtrix. Right (d): the orresponding sttering spetr of, nd. (solid yn line) is the sttering spetr of protein ggregte in the imge nd is rod nd low in intensity. (solid mgent line) nd (green solid line) re sttering spetr (shrp nd high in intensity) of nnoprtiles in the ggregted protein mtrix in the imge nd, respetively. The differene etween nd in the sttering pek position my e due to the surrounding environment. The prtiles from whih spetr re olleted re lelled. 9

10 Sttering intensity (.u.) Eletroni Supplementry Informtion 9C e 3 2 d d Wvelength (nm) Fig. S8C Sttering from vesiles were oserved in the ontrol untreted ells. Left: Imges,, nd d re four seleted re imges from the topmost imge of Figure 6C. Right (e): Corresponding sttering spetr of vesiles in,, nd d. Here the spetr re rod nd lesser in intensity unlike those of plsmoni nnoprtiles whih is key ftor to distinguish nnoprtiles from vesiles. The vesiles from whih spetr re olleted re mrked. 1

11 Sttering intensity (.u.) Eletroni Supplementry Informtion 9D e 8 Cells treted for 12 h with AuNP 4 d Wvelength (nm) d Fig. S9D. Sttering imges nd spetr of prtiles uptken y AuNP 4 treted ells. Left Side: Imges,, nd d re seleted re imges from the middle imge of Figure 6C, showing the presene of sttering from AuNPs. Right (e): Corresponding sttering spetr of AuNPs in the right side imges (solid lue line representing AuNPs in imge ), (solid red line representing AuNPs in imge ), (solid green line representing AuNPs in imge ) nd d (solid mgent line representing AuNPs in imge d), respetively. The prtiles from whih spetr re olleted re mrked. 11

12 Vinlstin treted A549 ells Eletroni Supplementry Informtion 1 α-tuulin TRITC DAPI Merged Fig. S1 Positive ontrol: Vinlstin (5 nm) treted A549 ells for 24 hours showing mirotuule dmge. Upper pnel imges show ontrol ells (non-treted). Lower pnel imges show vinlstin treted ells. Nuleus is stined with DAPI (lue) nd mirotuule is stined with nti-tuulin ntiody onjugted with TRITC (red). Sle r is 1 µm. 12

13 % of Cell viility % of Cell viility % of Cell viility % of Cell viility % of Cell Viility % of Cell viility Eletroni Supplementry Informtion 11 A pm AuNP pm AuNP 2 25 pm AuNP pm AuNP pm AuNP 4 25 pm AuNP pm AuNP pm AuNP 6 25 pm AuNP 6 Inution of AuNP 2 for 24 h Inution of AuNP 4 for 24 h Inution of AuNP 6 for 24h B pm AuNP pm AuNP 2 25 pm AuNP pm AuNP pm AuNP 4 25 pm AuNP pm AuNP pm AuNP 6 25 pm AuNP 6 Inution of AuNP 2 for 48 h Inution of AuNP 4 for 48h Inution of AuNP 6 for 48 h Fig. S11 A) Br digrm showing % of ell viility for the three different sized AuNPs t different onentrtions t 24 h. B) Br digrm showing % of ell viility for the three different sized AuNPs t different onentrtions t 48 h 13

14 Cell Viility (%) Eletroni Supplementry Informtion 12 MCF-7 ells Clulted IC 5 = pm AuNP 4 (pm) Fig. S12 Cell viility ssy of MCF-7 fter 72 h tretment with AuNP 4. The lulted IC 5 vlue ws 46.4±1.9 pm. Eletroni Supplementry Informtion 13 A B 12.5 pm C D 25. pm 5. pm Fig. S13 Oservtion of similr effet of AuNPs interting with MCF-7 ells. The ells were stined with TRITC ginst nti-α-tuulin ntiody. (A) MF7 ells, not treted with AuNPs. (B) Treted with 12.5 pm AuNP 4, (C) treted with 25. pm AuNP 4 nd (D) treted with 5. pm AuNP 4. Cellulr mirotuule struture ws monitored fter 72 h of inution. 14

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