Micellar stability in biological media dictates internalization in living cells

Transcription

1 Micellar stability in biological media dictates internalization in living cells Natalia Feiner-Gracia, Marina Buzhor,, Edgar Fuentes, ílvia Pujals, Roey J. Amir*,,,# and Lorenzo Albertazzi*, Contents 1. Experimental section Instrumentation Materials Methods upporting Figures References

2 1. Experimental section 1.1 Instrumentation HPLC: All measurements were recorded on a Waters Alliance e2695 separations module equipped with a Waters 2998 photodiode array detector. All solvents were purchased from Bio-Lab Chemicals and were used as received. All solvents are HPLC grade. 1 H and 13 C NMR: spectra were recorded on Bruker Avance I and Avance III 400MHz spectrometers. Chemical shifts are reported in ppm and referenced to the solvent. The molecular weights of the PEG-dendron hybrids were determined by comparison of the areas of the peaks corresponding to the PEG block (3.63 ppm) and the protons peaks of the dendrons. GPC: All measurements were recorded on Viscotek GPCmax by Malvern using refractive index detector and PEG standards (purchased from igma-aldrich) were used for calibration. DMF + 25mM NH 4 Ac was used as mobile phase. Infrared spectra: All measurements were recorded on a Bruker Tensor 27 equipped with a platinum ATR diamond. Absorbance and fluorescence spectra (including CMC measurements): pectra were recorded on an Agilent Technologies Cary Eclipse Fluorescence pectrometer using quartz cuvettes or on TECAN Infinite M200Pro plate reader device. MALDI-TF M: Analysis was conducted on a Bruker AutoFlex peed MALDI-TF- TF M (Germany). DHB matrix was used. DL: All measurements were recorded on a VAC-3 Particle ize Analyzer. Confocal imaging: All images were recorded using Zeiss LM780 pectral Confocal Microscope. 1.2 Materials Poly (Ethylene Glycol) methyl ether 5kDa, 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), Esterase from porcine liver (PLE), Allyl bromide (99%), N,N'-dicyclohexylcarbodiimide (DCC, 99%), Fmoc-Lys(Boc)-H (98%), ctanoic acid (98%), Undecanoic acid (98%) and ephadex LH20 were purchased from igma-aldrich. Cystamine hydrochloride (98%) and DIPEA were purchased from Merck. -(Benzotriazol-1-yl)-N,N,N,N -tetramethyluronium hexafluorophosphate (HBTU, 99.9%) was purchased from Chem-Impex. Trifluoroacetic acid (TFA) and 2- Mercaptoethanol (99%) were purchased from Alfa Aesar and phenyl acetic acid was purchased from Fluka. odium hydroxide and all solvents were purchased from Bio-Lab and were used as received. Deuterated solvents for NMR were purchased from Cambridge Isotope Laboratories, Inc. Bovine erum Albumin (lyophilized powder 96%) and Triton X-100 were purchased from igma-aldrich. Fetal Bovine erum was purchased from Thermofisher, it was stored at 22 ºC and was only defreeze before used. Cy-5 was purchased from Lumiprobe. PrestoBlue was purchased from Thermofisher. 2

3 m m 1.3 Methods ynthesis H H m Me n N H PEG-Lys(Boc)-dendron-(H) 4 (Hybrid 5a) H N HN Boc H H H m DCC, DMAP (cat) DCM Me n N H PEG-Lys(Boc)-dendron-(Aliphatic) 4 H N HN Boc m Hybrid 2a: m=6 Hybrid 1a: m=9 m m 1. TFA:DCM (1:1) (Boc deprotection) 2. Labeling moiety HBTU, DIPEA in DMF:DCM Me n N H PEG-Lys(Labeled)-dendron-(Aliphatic) 4 H N HN m Labeling Moiety m Figure 1. General procedure for preparation of compounds 1a, 2a and labeled aliphatic hybrids. General procedure for Me-PEG 5kDa -Lys(Boc)-dendron-(Aliphatic) 4 : Me-PEG 5kDa -Lys(Boc)-dendron-(H) 4 1 (5a) was dissolved in DCM (1mL, per 0.1g). Aliphatic acid (20 eq.) were added. The flask was cooled to 0 C followed by the addition of DCC (20 eq.) and DMAP (0.1eq.) dissolved in DCM (1mL). The reaction was stirred for 1 hour at room temperature. The crude mixture was filtered and the organic solution was evaporated to dryness. The residue was re-dissolved in DCM (5mL per 1g) and the product was precipitated by the drop wise addition of Ether (50mL per 1g). The precipitate underwent centrifugation and was separated from the organic solvent. The precipitate was collected and residual of solvents were evaporated under vacuum. The residue was dissolved in MeH and loaded on a MeH based LH20 EC column. The fractions that contained the product were unified and the MeH was evaporated in vacuum to yield an oily residue. In order to facilitate the removal of residual MeH and solidification of the product, the oily residue was re-dissolved in DCM (1mL) followed by addition of Hexane (3mL). DCM and Hexane were evaporated to dryness and the obtained solid was dried under high vacuum. 3

4 Hybrid 2a Me-PEG 5kDa -Lys(Boc)-dendron-(ct) 4 245mg (0.042mmol) of Me-PEG 5kDa -Lys(Boc)-dendron-(H) 4 (5a) and ctanoic acid (0.830mmol, 20eq.) were reacted according to the general procedure and the product was obtained as an off-whit solid (240mg, 90% yield). 1 H NMR (400 MHz, Chloroform-d): δ (m, 3H, -CH-NH-C-Ar- + Ar-H), (m, 2H, CH 2 -NH-C-CH- + Ar-H), 4.66 (m, 1H, -NH-Boc), 4.53 (q, J = 7.5 Hz, 1H, -C-CH-NH-), (m, 12H, -CH 2 --C- + -Ar--CH 2 -), (m, 514H, PEG backbone), 3.34 (s, 3H, CH 3 --PEG), 3.16 (q, J = 6.1 Hz, 2H, -CH--), 3.06 (m, 2H, Boc-NH-CH 2 -), (m, 8H, - CH-CH CH--CH 2 -), (m, 4H, -CH-CH 2 --CH 2 -), 2.59 (dt, J = 22.0, 7.0 Hz, 3H, - CH 2 --CH 2 -), 2.27 (td, J = 7.5, 1.9 Hz, 8H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 ), (m, 4H, -- CH 2 -CH 2 -CH Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), 1.59 (m, 10H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 + Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), 1.37 (m, 11H, Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 -CH + Boc), (m, 34H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 ), (m, 12H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 ). 13 C NMR (101 MHz, CDCl3): δ 173.1, 171.0, 166.3, 159.1, 155.7, 149.1, 135.6, 105.8, 104.3, 71.5, 70.5, 69.8, 69.7, 69.3, 68.9, 62.9, 62.6, 58.5, 53.0, 45.1, 39.6, 38.2, 34.4, 33.7, 31.6, 31.2, 31.1, 29.9, 29.2, 29.1, 28.6, 28.5, 28.4, 27.9, 27.9, 24.4, 22.4, 22.1, FT-IR, ν (cm -1 ): 2883, 1734, 1594, 1466, 1457, 1360, 1341, 1279, 1240, 1147, 1102, 1060, 960, 842; GPC: Mn = 7.0kDa, PDI = Expected Mn = 6.4kDa. Figure 2. 1 H-NMR spectra of hybrid 2a. Hybrid 1a Me-PEG 5kDa -Lys(Boc)-dendron-(Und) 4 267mg (0.045mmol) of Me-PEG 5kDa -Lys(Boc)-dendron-(H) 4 (5a) and Undecanoic acid (0.910mmol, 20eq.) were reacted according to the general procedure and the product was obtained as an off-whit solid (268mg, 90% yield). 4

5 1 H NMR (400 MHz, Chloroform-d): δ (m, 3H, -CH-NH-C-Ar- + Ar-H), (m, 2H, CH 2 -NH-C-CH- + Ar-H), 4.66 (m, 1H, -NH-Boc), 4.53 (q, J = 7.6 Hz, 1H, -C-CH-NH-), (m, 12H, -CH 2 --C- + -Ar--CH 2 -), (m, 498H, PEG backbone ), 3.34 (s, 3H, CH 3 --PEG), 3.17 (q, J = 5.9, 2H, -CH--), 3.06 (q, J = 6.7 Hz, 2H, Boc-NH-CH 2 -), (m, 8H, -CH-CH CH--CH 2 -), (m, 4H, -CH-CH 2 --CH 2 -), 2.59 (dt, J = 21.9, 7.0 Hz, 4H, -CH 2 --CH 2 -), 2.27 (td, J = 7.5, 2.0 Hz, 8H, --C-CH 2 -CH 2 -(CH 2 ) 7 -CH 3 ), (m, 4H, --CH 2 -CH 2 -CH Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), (m, 10H, --C-CH 2 - CH 2 -(CH 2 ) 7 -CH 3 + Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), 1.38 (m, 11H, Boc-NH-CH 2 -CH 2 -CH 2 -CH 2 - CH + Boc), 1.22 (m, 59H, --C-CH 2 -CH 2 -(CH 2 ) 7 -CH 3 ), 0.84 (t, J = 6.9 Hz, 12H, --C-CH 2 - CH 2 -(CH 2 ) 7 -CH 3 ). 13 C NMR (101 MHz, CDCl 3 ): δ 173.1, 171.0, 166.3, 159.1, 155.6, 135.6, 105.8, 104.3, 71.4, 70.3, 69.87, 69.7, 69.3, 68.9, 62.9, 62.6, 58.5, 53.0, 45.0, 39.6, 38.2, 34.4, 33.7, 31.6, 31.4, 31.1, 31.0, 29.9, 29.2, 29.1, 29.0, 28.9, 28.8, 28.7, 28.6, 28.0, 27.8, 24.4, 22.4, 22.2, FT- IR, ν (cm -1 ): 2884, 1735, 1594, 1466, 1360, 1341, 1279, 1240, 1147, 1102, 1060, 960, 842; GPC: Mn = 7.3kDa, PDI = Expected Mn = 6.5kDa. Figure 3. 1 H-NMR spectra of hybrid 1a. General procedure for Me-PEG 5kDa -Lys(Labeled)-dendron-(Aliphatic) 4 Me-PEG 5kDa -Lys(Boc)-dendron-(Aliphatic) 4 (1a or 2a) was dissolved in DCM (1mL) and TFA was added (1mL). After 30 minutes the solution was evaporated to dryness and dried in vacuum. The labeling moiety with a carboxylic acid functional group (2eq.) and HBTU (2eq.) were dissolved in DCM:DMF 1:1 (1mL) followed by addition of DIPEA (20eq.). The solution was added to the deprotected hybrid (Me-PEG 5kDa -Lys(NH 2 )-dendron-(aliphatic) 4 ) dissolved in DCM (1mL, per 0.1g). The reaction was stirred for 1 hour in room temperature. The crude mixture was concentrated under vacuum and loaded on a MeH based LH20 EC column. The fractions that contained the product were unified and the DCM and MeH were evaporated in vacuum to yield an oily residue. 5

6 In order to facilitate the removal of residual MeH and solidification of the product, the oily residue was re-dissolved in DCM (1mL) followed by addition of Hexane (3mL). DCM and Hexane were evaporated to dryness and the obtained solid was dried under high vacuum. m m Figure 4. General procedure for preparation of Coumarin labeled aliphatic hybrids - 1, 2. Hybrid 2 Me-PEG 5kDa -Lys(Coumarin)-dendron-(ct) 4 104mg (0.016mmol) of Me-PEG 5kDa -Lys(Boc)-dendron-(ct) 4 (2a) were deprotected to yield Me-PEG 5kDa -Lys(NH 2 )-dendron-(ct) 4 that were reacted with 7-(diethylamino)-3-carboxy Coumarin [2] (0.032mmol, 2eq.) according to the general procedure and the product was obtained as a yellow solid (98mg, 92% yield). 1 H NMR (400 MHz, Chloroform-d) δ 8.82 (m, 1H, -CH 2 -CH 2 -NH-C-), 8.52 (s, 1H, Ar-H), 7.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.19 (d, J = 6.8 Hz, 1H, -CH-NH-C-Ar-), 7.02 (m, 2H, Ar-H), 6.78 (m, 1H, -CH 2 -NH-C-CH-), (m, 2H, Ar-H), 6.44 (s, 1H, Ar-H), 4.51 (m, 1H, -C-CH-NH-), (m, 12H, -CH 2 --C- + -Ar--CH 2 -), (m, 535H, PEG backbone), 3.33 (s, 3H, CH 3 --PEG), 3.16 (t, J = 5.9 Hz, 2H, -CH--), (m, 12H, -CH-CH 2 --CH CH-- CH 2 -), 2.58 (dt, J = 25.5, 6.9 Hz, 3H, -CH 2 --CH 2 -), 2.26 (t, J = 7.7 Hz, 8H, --C-CH 2 -CH 2 - (CH 2 ) 4 -CH 3 ), (m, 4H, --CH 2 -CH 2 -CH NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), (m, 12H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 + -NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), 1.22 (q, J = 7.0, 6.4 Hz, 44H, --C-CH 2 -CH 2 -(CH 2 ) 4 -CH 3 + -N-CH 2 -CH 3 ), (m, 13H, --C-CH 2 -CH 2 -(CH 2 ) 4 - CH 3 ). 13 C NMR (101 MHz, CDCl 3 ): δ 173.7, 171.7, 167.1, 163.5, 162.8, 159.5, 157.6, 152.6, 148.2, 136.3, 110.2, 108.4, 96.6, 71.8, 70.6, 69.5, 63.4, 63.1, 45.5, 45.1, 34.3, 31.7, 30.4, 29.6, 29.2, 29.0, 25.0, 22.7, 14.2, FT-IR, ν (cm -1 ): 2883, 1734, 1699, 1648, 1617, 1585, 1541, 1514, 1466, 1359, 1341, 1279, 1240, 1147, 1100, 1060, 960, 812; GPC: Mn = 7.0kDa, PDI = Expected Mn = 6.5kDa. MALDI-TF M: molecular ion centered at 6.5kDa. 6

7 Me n N H HN HN N f1 (ppm) Figure 5. 1 H-NMR spectra of hybrid 2. Figure 6. MALDI spectrum of hybrid 2. Hybrid 1 Me-PEG 5kDa -Lys(Coumarin)-dendron-(Und) 4 81mg (0.012mmol) of Me-PEG 5kDa -Lys(Boc)-dendron-(Und) 4 (1a) were deprotected to yield Me- PEG 5kDa -Lys(NH 2 )-dendron-(und) 4 that were reacted with 7-(diethylamino)-3-carboxy coumarin (0.024mmol, 2eq.) according to the general procedure and the product was obtained as a yellow solid (81mg, quantitative yield). 7

8 1 H NMR (400 MHz, Chloroform-d): δ 8.84 (t, J = 5.9 Hz, 1H, -CH 2 -CH 2 -NH-C-), 8.55 (s, 1H, Ar- H), 7.37 (dt, J = 9.0, 1.2 Hz, 1H, Ar-H), 7.18 (d, J = 7.8 Hz, 1H, -CH-NH-C-Ar-), 7.02 (d, J = 2.3 Hz, 2H, Ar-H), 6.78 (t, J = 5.7 Hz, 1H, -CH 2 -NH-C-CH-), (m, 2H, Ar-H), 6.47 (d, J = 2.3 Hz, 1H, Ar-H), 4.54 (t, J = 6.5 Hz, 1H, -C-CH-NH-), (m, 11H, -CH 2 --C- + -Ar- -CH 2 -), (m, 522H, PEG backbone), 3.35 (s, 3H, CH 3 --PEG), (m, 2H, - CH--), (m, 8H, -CH-CH CH--CH 2 -), (m, 4H, -CH-CH 2 --CH 2 -), 2.61 (dt, J = 26.0, 7.0 Hz, 4H, -CH 2 --CH 2 -), (m, 9H, --C-CH 2 -CH 2 -(CH 2 ) 7 -CH 3 ), (m, 4H, --CH 2 -CH 2 -CH NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), (m, 11H, -- C-CH 2 -CH 2 -(CH 2 ) 7 -CH 3 + -NH-CH 2 -CH 2 -CH 2 -CH 2 -CH-), 1.23 (q, J = 4.3, 3.3 Hz, 63H, --C- CH 2 -CH 2 -(CH 2 ) 7 -CH 3 + -N-CH 2 -CH 3 ), 0.85 (t, J = 6.9 Hz, 12H, --C-CH 2 -CH 2 -(CH 2 ) 7 -CH 3 ). 13 C NMR (101 MHz, CDCl 3 ): δ 173.1, 171.7, 171.1, 166.5, 162.9, 162.2, 159.0, 157.1, 152.0, 147.5, 135.8, 130.6, 129.2, 109.5, 107.9, 105.9, 104.5, 96.1, 81.6, 71.5, 70.1, 69.3, 69.0, 68.8, 62.9, 62.6, 58.5, 53.2, 50.5, 49.1, 45.1, 44.6, 38.2, 34.4, 33.7, 31.4, 31.1, 29.9, 29.2, 29.1, 29.0, 28.9, 28.7, 27.8, 24.4, 22.6, 22.2, 13.6, 12.0, 0.5. FT-IR, ν (cm -1 ): 2884, 1735, 1699, 1647, 1617, 1585, 1541, 1514, 1466, 1359, 1341, 1279, 1240, 1147, 1102, 1060, 960, 842; GPC: Mn = 7.1kDa, PDI = Expected Mn = 6.7kDa. MALDI-TF M: molecular ion centered at 6.7kDa. Figure 7. 1 H-NMR spectra of hybrid 1. 8

9 Figure 8. MALDI spectrum of hybrid 1. Characterization of hybrids Gel permeation chromatography (GPC): Hybrids were dissolved in mobile phase (DMF + 25mM NH 4 Ac) to give final concentrations of 10mg/ml. olution was filtered through a 0.22µm PTFE syringe filter. The columns used were 2 x P GRAM 1000Å + P GRAM 30Å at 50ºC. 50 µl of mobile phase were injected at a flow rate of 0.5ml/min. Refractive index detector was set at 50 C (Figures 9 and 10). Critical micelle concentration (CMC) measurements: Diluent solution was prepared as followed; 10 ml Phosphate buffer solution (ph 7.4), 4.5µL of Nile red stock solution (0.88mg/mL in Ethanol) were mixed to give a final concentration of 1.25µM. To measure CMC for compounds 1 and 2 a 500µM solution of each hybrid was prepared in diluent and sonicated for 15 minutes. This solution was repeatedly diluted by a factor of 1.5 with diluent. 150µL of each solution were loaded onto a 96 wells plate. The fluorescence emission intensity was scanned for each well, by exciting at 550 nm and measuring the emission spectra from 580 to 800nm. Maximum emission intensity was plotted vs. hybrid concentration in order to determine the CMC. All measurements were repeated 3 times (Figure 11). Dynamic Light cattering (DL): Hybrids 1-5 were separately dissolved in phosphate buffer (ph 7.4) to give final concentration of 160µM. The olutions were sonicated for 15 minutes and filtered through a 0.22µm nylon syringe filter. Measurements were performed (t=0 before addition of enzyme). All measurements were repeated 3 times (Figure 12). Transmission Electron Microscopy (TEM): Hybrid 1 was prepared at a concentration of 160 µm and 0.5 µm in MiliQ water and let stabilize for 72 hours. Then, samples were deposited onto a glow discharged C-only grids and negative staining was performed using uranyl acetate at 2%. All electron micrographs 9

10 were obtained with a Jeol JEM 1010 MT electron microscope (Japan) operating at 80 kv. Images were obtained on a CCD camera Megaview III (II), Münster, Germany. Comparing HPLC and fluorescence monitoring of enzymatic degradation: For the HPLC analysis of degradation a Phenomenex, Aeris WIDEPRE, C4, 150x4.6mm, 3.6µm column was used at a temperature of 30ºC, and the UV detector set at 256nm, 2Hz detection rate. Three mobile phase were used i) solution A: 0.1% HCl 4 in H 2 :Acetonitrile 95:5 V/V, ii) solution B: 0.1% HCl 4 in H 2 :Acetonitrile 5:95 V/V and iii) solution C: THF. A gradient program in time was used were percentage of solutions A/B/C was changed from 95/0/5 at 1 min to 0/95/5 at 20 min and from 0/95/5 at 23 minutes to 95/0/5 at 23.1 minutes. For HPLC monitoring hybrids 1 and 2 were separately dissolved in phosphate buffer to give a concentration of 160µM. Each solution was sonicated for 15 minutes. 30µL of hybrid solution were injected to the HPLC as t=0 injection. PLE enzyme stock solution (36.0µM or 72.0µM in phosphate buffer ph 7.4) were added to each solution of the tested hybrid (160µM) and mixed for 10 seconds (vortex mixer) to give final PLE concentration of 685nM or 2µM. Enzymatic degradation was monitored by repeating 30µL injections from the same vail over 15 hours. All measurements were repeated 3 times (Figures 17-20). The fluorescence monitoring of micelle disassembly rate by enzymes was performed using an Agilent Technologies Cary Eclipse Fluorescence pectrophotometer. Hybrids 1 and 2 were prepared the same as for HPLC measurements (160µM, PB). For each hybrid 600µL were accurately transferred to a quartz cuvette. A fluorescence emission scan was performed (t=0). For enzymatic degradation 600µL were transferred to quarts cuvette and mixed with the enzymes at either 685nM or 2µM final concentration. Hybrids were excited at 420 nm and emission scan was measured from 440 to 700 nm at a scan rate of 590 nm/min, all measurements were carried out at 37ºC (Figures 13 and 14). Repeating fluorescence scans were performed either every 30 minutes or 2 hours for about 15 hours or 160 hours respectively. All measurements were repeated 3 times (Figures 13-16).. Micelle blood proteins interaction Interaction of monomer and blood proteins: Hybrid 4 was prepared at 2 µm in filtered Phosphate Buffered aline (PB) 0.16M, the fluorescence emission spectrum of the micelles was monitor using Tecan infinite M200Pro microplate reader. The measured emission wavelength ranged from 450 nm to 650 nm, using an excitation wavelength of 420 nm. BA was added at a final concentration of 10 mg/ml, modifying the micelle concentration to 1,82 µm. The Fluorescence emission spectrum was measured for 9 cycles every 40 s. Förster Resonance Energy Transfer (FRET) was used to measure the interactions between the dye of the hybrids (coumarin) and Cy5 conjugated. Micelles at a final concentration of 145,5 µm were mixed with BA (10% v/v Cy5 labelled) at a final concentration of 5.5 mg/ml. The sample was excited at 420 nm wavelength (coumarin excitation, donor), and the fluorescence emission spectra was measured from 450 nm to 750 nm to include Cy5 emission. The spectra was measured before the addition of BA and 3 more times every 15 minutes after the addition. 10

11 upramolecular stability BA titration: All hybrids were prepared at a final concentration of 40 µm (in PB). BA was added at increasing concentrations from 0 to 1mM in PB. Emission fluorescence spectra of each condition was measured using Tecan infinite M200Pro microplate reader. amples were excited at 420 nm and emission spectra was collected from 450 nm to 650 nm. tability in time: To perform studies on stability of micelles in time, hybrids solutions were prepared at a concentration of 160 µm of monomer (in PB). Each hybrid was mixed with BA at a final concentration of 10 mg/ml in PB, FB at a final concentration of 10% v/v or PB as a control. Then, Tecan infinite M200Pro microplate reader was used to measure emission fluorescence spectra in time every hour, for 12 hours. amples were excited at 420 nm and emission spectra was collected from 450 nm to 650 nm. Resistance to dilution: To determine the stability of micelles against dilution each hybrid was prepared at a concentration of 160 µm (in PB). Then, it was sequentially diluted either in PB or FB up to 78 nm. Then, Tecan infinite M200Pro microplate reader was used to measure emission fluorescence spectra every 15 min for 4 hours. amples were excited at 420 nm and emission spectra was collected from 450 nm to 650 nm. Enzymatic response The enzymatic response of the micelles was studied in presence of BA and FB comparing to control conditions without proteins. Each hybrid was prepared at a concentration of 160 µm (in PB). Then, each hybrid was mixed with BA at a final concentration of 10 mg/ml, FB at a final concentration of 10% v/v or PB as a control and let stabilize for 1 hour. Fluorescence emission spectra were measured for each condition before and after the addition of the protein. Then, PLE at a concentration of 5 µm in the ester responsive micelles and PGA at a concentration of 50 µm in the amidase responsive sample were added to a final concentration of 270 nm and 910 nm respectively. Following by the measurement in time of the spectrum for 15 hours. The enzyme concentration was selected considering the concentrations used in previous works 1. Hybrids- cell interactions Cytotoxicity: The cytotoxicity of all the samples was evaluated with PrestoBlue assay on HeLa cells culture. HeLa cells were seeded in a 96-well plate at a density of cells/well and cultured overnight at 37ºC and 5% C 2. Hybrids were added to the cells in full medium to a concentration of 160 µm, the same volume of PB was added to the negative controls, Triton X-100 0,1 %v/v was added to the positive controls. The hybrids were incubated for 24 hours at 37º and 5% C2.Then, PrestoBlue reactive was added at 10 % v/v. The emitted fluorescence at 600 nm of each well was measured after exciting at 550 nm.the wells at the boundary remained untested with micelles. Each sample had 3 replications. Fluorescent signal was normalized with negative and positive controls to 0 and 100% respectively. 11

12 Internalization: HeLa cells were seeded in a 8-wells Nunc Lab-Tek II Chamber lide (ThermoFisher) and cultured overnight at 37ºC and 5% C 2, cells/well. Hybrids at a final concentration of 160 µm in complete medium supplemented with 20 mm Hepes were added and live imaging of cells at a 37ºC was performed in a Zeiss LM780 pectral Confocal Microscope. Hybrids were excited using 405 nm laser and emission spectra was collected after 10 and 30 minutes of incubation using two approaches. i) Two different PMT detectors were used to collect emission of monomer and micelle separately and simultaneous, windows of detection were set as following, monomer nm and micelle nm. ii) pectral images were acquired using a 32 PMT GaAsP array detector, with allowed to collect a lambda stack with steps of 9 nm from 411 nm to 695 nm. Ratiometric images were obtained from dividing the monomer image by the micelle image, after applying a mask to each image were noise was removed. Experiments were localization of micelles was studied micelles were incubated for 1 hour and then a cellular compartment tracker was added, LysoTracker was used at a concentration of 1nM and ER-Tracker at a concentration of 40 nm. Confocal images were acquired by exciting hybrids with 405 nm laser and Red Tracker with 561 nm laser. 12

13 2 upporting Figures Figure 9. GPC traces of mpeg 5kDa -Lys(Boc)-Fmoc (blue), mpeg 5kDa -Lys(Boc)-diyne (red), mpeg 5kDa -Lys(Boc)-(H) 4 (green)- hybrid 5a, mpeg 5kDa -Lys(Boc)-(ct) 4 (purple)- hybrid 2a, mpeg 5kDa -Lys(Coum)-(ct) 4 (light blue) hybrid 2. (*The previously synthesized hybrids were analyzed against hybrid 2 1 ). Figure 10. GPC traces of mpeg 5kDa -Lys(Boc)-Fmoc (blue), mpeg 5kDa -Lys(Boc)-diyne (red), mpeg 5kDa -Lys(Boc)-(H) 4 (green)- hybrid 5a, mpeg 5kDa -Lys(Boc)-(Und) 4 (orange)- hybrid 1a, mpeg 5kDa -Lys(Coum)-(Und) 4 (light blue) hybrid 1. (*The previously synthesized hybrids were analyzed against hybrid 1 1 ). 13

14 Normalized RFU 40 Hybrid 1 mpeg 5kDa -Lys(Coum)-(Und) 4 CMC = 2 ± 1 µm Hybrid 2 mpeg 5kDa -Lys(Coum)-(ct) 4 CMC = 5 ± 1 µm Conc. (µm) Figure 11. CMC measurements of hybrids 1 and 2. Hybrid 1 36 ± 7 nm 0.2 Hybrid 2 28 ± 5 nm Hybrid 3 24 ± 5 nm Volume (A.U.) 0.1 Hybrid 4 22 ± 7 nm Hybrid 5 3 ± 1 nm Dh (nm) Figure 12. DL measurements of hybrids 1-5 in phosphate buffer 160µM of hybrid. 14

15 Figure 13. Transmission electron microscopy images of hybrid 1 at 160 µm and 0.5 µm after 72 hours stabilization. The images showed that at 0.5 µm there are still some micelles assembled. t=0 hrs (addition of 685nM PLE ) 800 t= 15 hrs 600 RFU 400 (A.U.) λ (nm) Figure 14. Time-dependent fluorescence spectra of hybrid 2 (160 µm) after the addition of PLE (685 nm) after 15 hours. 15

16 800 t= 168 hrs t=0 hrs (addition of 2µM PLE ) 600 RFU (A.U.) λ (nm) Figure 15. Time-dependent fluorescence spectra of hybrid 2 (160 µm) after the addition of PLE (2 µm) after 168 hours t= 15 hrs t=0 hrs (addition of 685nM PLE ) RFU (A.U.) λ (nm) Figure 16. Time-dependent fluorescence spectra of hybrid 1 (160 µm) after the addition of PLE (685 nm) after 15 hours. 16

17 800 t=0 hrs (addition of 2µM PLE ) t= 160 hrs 600 RFU (A.U.) λ (nm) Figure 17. Time-dependent fluorescence spectra of hybrid 1 (160 µm) after the addition of PLE (2 µm) after 160 hours. Figure 18. HPLC monitoring of micelle degradation in presence of 685nM PLE enzyme for hybrid 2 (160 µm) over 15 hours. 17

18 Figure 19. HPLC monitoring of micelle degradation in presence of 2µM PLE enzyme for hybrid 2 over (160µM) 165 hours. Figure 20. HPLC monitoring of micelle degradation in presence of 685nM PLE enzyme for hybrid 1 (160 µm) over 15 hours. 18

19 Figure 21. HPLC monitoring of micelle degradation in presence of 2µM PLE enzyme for hybrid 1 (160µM) over 160 hours. 100 Fluorescence(λ = 540 nm) without PLE Mol % Hybrid 2(HPLC) After addition of 685 nm PLE Fluorescence (λ = 540 nm) After addition of 685 nm PLE RFU (%) Time (Hour) 0 Figure 22. verlays of the change in fluorescence (λ=540nm) and HPLC analysis of the enzymatic degradation (685nM PLE) of hybrid 2 (160µM). 19

20 Mol % Fluorescence (λ = 480 nm) After addition of 2 µm PLE Hybrid 2(HPLC) After addition of 2 µm PLE Hybrid 5 (HPLC) After additiob of 2 µm PLE Fluorescence (λ = 540 nm) After addition of 2 µm PLE Time (Hour) RFU (%) Figure 23. verlays of the change in fluorescence (λ=540nm) and HPLC analysis of the enzymatic degradation (2µM PLE) of hybrid 2 (160µM) Fluorescence without PLE (λ = 540 nm) 80 Mol % Fluorescence (λ = 540 nm) After addition of 685 nm PLE Hybrid 1(HPLC) After additiob of 685 nm PLE RFU (%) Time (Hour) 0 Figure 24. verlays of the change in fluorescence (λ=540nm) and HPLC analysis of the enzymatic degradation (685nM PLE) of hybrid 1 (160µM). 20

21 Fluorescence without PLE (λ = 540 nm) Fluorescence (λ = 540 nm) After addition of 2 µm PLE Mol % 40 Hybrid 1(HPLC) After additiob of 2 µm PLE 40 RFU (%) Time (Hour) 0 Figure 25. verlays of the change in fluorescence (λ=540nm) and HPLC analysis of the enzymatic degradation (2µM PLE) of hybrid 1 (160µM). Fluorescence (A.U.) Hybrid 5 Hybrid 5 + BA Wavelength (nm) Figure 26. Interaction of monomer with BA. Hybrid 5 at a concentration of 160 µm was incubated with BA at a concentration of 400 µm. An increase in the emission intensity can be observed attributed to the solvathocromic effect of coumarin when interact with the monomer. 21

22 Figure 27. Controls of FRET studies between micelle and BA. Emitted fluorescence of hybrid 3 (160 µm) without BA and the emitted fluorescence of BA-Cy5 at 10 mg/ml, after excitation at 420 nm (usual hybrid excitation). The spectra show that the appearance of a peak at 675 nm when the hybrid is mixed with the BA-Cy5 is because the interactions of the assembled hybrid and the BA- Cy5 and not just because of the presence of Cy5. Figure 28. pectrum of hybrid 4 at increasing concentrations of BA, from 0 to 1000 µm (a) pectrum of all concentrations (b) pectrum of BA from 5 to 400 µm which show around 100 µm micelle start to disassemble, not only monomer peak (470 nm) increase but micelle peak (at 560 nm) start to decrease. 22

23 Figure 29. Ratio of emitted fluorescence of monomer versus micelle at different time intervals in PB (left), 10 mg/ml BA (center) and 10% v/v FB (right) for all the hybrids. Figure 30. tability as a function of time in PB and 10% v/v FB at 37ºC shows a smooth reduction of the stability in every condition compared to the stability at 25ºC. The graphs represent the ratio of monomer emitted fluorescence vs micelle emitted fluorescence (a) Hybrid 1 (b) Hybrid 2 (c) Hybrid 3 and (d) Hybrid 4. 23

24 Figure 31. Emission fluorescence spectra as a function of dilution in PB from 160 µm to 78 nm, the left and right graphs show different dilution steps. (a) Hybrid 1 (b) Hybrid 2 (c) Hybrid 3 and (d) Hybrid 4. Values lower than 300 A.U. are under the limit of detection. 24

25 Figure 32. Emission fluorescence spectra as a function of dilution in 10% v/v FB from 160 µm to 78 nm, the left and right graphs show different dilution steps. (a) Hybrid 1 (b) Hybrid 2 (c) Hybrid 3 and (d) Hybrid 4. Values lower than 300 A.U. are under the limit of detection. 25

26 Figure 33. tability of the micelles upon dilution (in 10% v/v FB) showed hybrid 4 and hybrid 3 disassembly occurs immediately after dilution but hybrid 2 and hybrid 1 disassemble is much slower. Graphs represent the emission fluorescence spectra at different time points after dilution from 0h to 12h. (a) Hybrid 4 at 80 µm in FB (b) Hybrid 3 at 40 µm in FB (c) Hybrid 2 at 80 µm and (c) Hybrid 1 at 80 µm 26

27 Figure 34. Cytotoxicity of micelles at a concentration of 160 µm of hybrid (in PB) in HeLa cells. Data has been normalized considering a positive and a negative control. 27

28 Figure 35. Confocal images of the localization of hybrid 4 (a and b) and hybrid 5 (c and d) in HeLa cell (green) after 1 hour incubation and addition of ERtracker (red). cale bar 5 µm. The intensity profile of both hybrid and ER tracker in the pink line of the images have been plotted normalized to be able to compare both dyes fluorescence. The graphs show similar profiles for ER and hybrid which indicates colocalization. 28

29 Figure 36. The intracellular location of Hybrid 4 and Hybrid 1 was studied after 1 hour of incubation in HeLa cells. Cells were washed and then ER or lysosomes were stained using Red ERtracker and Red Lysotracker respectively. cale bar 5µm. 29

30 Figure 37. Localization of hybrid 5 in HeLa cell after 1 hour incubation and addition of ERtracker or Lysotracker. cale bar 5 µm. 3 References 1. M. Buzhor, A. J. Harnoy, E. Tirosh, A. Barak, T. chwartz, R. J. Amir, Chem. Eur. J. 2015, 21, G. He, D. Guo, C. He, X. Zhang, X. Zhao, C.Duan, Angew. Chem. Int. Ed. 2009, 48,