Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells

Article

Reference

Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells

VOROBIEV, Vassily, et al.

Abstract

Molecular medical imaging is intended to increase the accuracy of diagnosis, particularly in cardiovascular and cancer-related diseases, where early detection could significantly increase the treatment success rate. In this study, we present mixed micelles formed from four building blocks as a magnetic resonance imaging targeted contrast agent for the detection of atheroma and cancer cells. The building blocks are a gadolinium-loaded DOTA ring responsible for contrast enhancement, a fibrin-specific CREKA pentapeptide responsible for targeting, a fluorescent dye and DSPE-PEG2000. The micelles were fully characterized in terms of their size, zeta potential, stability, relaxivity and toxicity. Target binding assays performed on fibrin clots were quantified by fluorescence and image signal intensities and proved the binding power. An additional internalization assay showed that the micelles were also designed to specifically enter into cancer cells. Overall, these multimodal mixed micelles represent a potential formulation for MRI molecular imaging of atheroma and cancer cells.

VOROBIEV, Vassily, et al. Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells. European Journal of Pharmaceutics and Biopharmaceutics, 2021, vol. 158, p. 347-358

DOI : 10.1016/j.ejpb.2020.11.017 PMID : 33271302

Available at:

http://archive-ouverte.unige.ch/unige:149070

Disclaimer: layout of this document may differ from the published version.

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1 SUPPLEMENTARY INFORMATION

Journal:

The European Journal of Pharmaceutics and Biopharmaceutics

https://www.journals.elsevier.com/european-journal-of-pharmaceutics-and-biopharmaceutics

Research paper Title:

Vascular-targeted micelles as a specific MRI contrast agent for molecular imaging of fibrin clots and cancer cells

Author names and affiliations:

Vassily Vorobiev^a,b, Souad Adriouach^a,b, Lindsey A Crowe^c, Sébastien Lenglet^d, Aurélien Thomas^e,f, Anne-Sophie Chauvin^g, Eric Allémann^a,b,

*

aSchool of Pharmaceutical Sciences, University of Geneva, 1211 Geneva, Switzerland

bInstitute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, 1211 Geneva, Switzerland

cDepartment of Radiology and Medical Informatics, University of Geneva, 1211 Geneva, Switzerland

dForensic Toxicology and Chemistry Unit, University Center for Legal Medicine, Geneva University Hospital, 1211 Geneva, Switzerland

eUnit of Toxicology, CURML, Lausanne University Hospital, Geneva University Hospitals, Switzerland

fFaculty of Biology and Medicine, University of Lausanne, 1015 Lausanne, Switzerland

gInstitut of Chemical Sciences and Engineering, Swiss Federal Institute of Technology of Lausanne, Route Cantonale, 1015 Lausanne, Switzerland

*

Professor Eric Allémann

Institute of Pharmaceutical Sciences of Western Switzerland CMU Bâtiment B – 8ème étage, B08.1713

Rue Michel-Servet 1, 1211 GENEVE 4 Switzerland

Email: Eric.Allemann@unige.ch Phone number: + 41223796148

2 Table of contents

Materials and Methods ... 3

Synthesis of Gd-DO3A-Tyr-(C18)2 ... 5

Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 1 ... 6

Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 2: ... 8

Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 3: ... 11

Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 4: ... 14

Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 5: ... 17

Synthesis of Gd-DO3A-Tyr-(C18)2 – Final step: Gadolinium loading ... 19

Synthesis of DSPE-PEG2000-CREKA (and CERAK) ... 22

Synthesis of DSPE-PEG2000-IR-774 – all steps ... 24

Synthesis of DSPE-PEG2000-IR-774 – Step 1: Compound 1 ... 25

Synthesis of DSPE-PEG2000-IR-774 – Step 2: final structure ... 28

Absorbance and emission spectrums of DSPE-PEG2000-IR-774 ... 29

Cell internalization and fluorescence imaging: cells selection images of Mixes-1,2,3 and Anti-Mixes- 1,2,3 – MCF-7 cell line ... 30

Cell internalization and fluorescence imaging: cells selection images of Mixes-1,2,3 and Anti-Mixes- 1,2,3 – MCF-10 cell line ... 31

Cell internalization and fluorescence imaging: fluorescence quantification and graphical presentation ... 32

References: ... 33

3 Materials and Methods

Reagents were obtained from Sigma, Acros, Frontier Scientific and ThermoFisher Scietific and used as obtained. Milli-Q (Millipore Corporation, USA) ultrapure water with resistivity of 18.3 MΩcm at 25 °C was used in all experiments. Thin layer chromatography (TLC) was performed with aluminium backed silica plates (Merck- Keiselgel 60 F254) with a suitable mobile phase and was visualized using a UV fluorescence lamp (254 and 366 nm). Flash chromatography was done using an automated PuriFlash® 4100 system from Interchim (Montlucon, France) equipped with a PDA detector (200 – 800 nm) and automated fraction collector using Interchim puriFlash® HP 30 μm silica columns. The elution profile was monitored using Flash Interchim software version 5.0x. Analytical UPLC was conducted using Macherey- Nagel EC50/2 Nucleodur Gravity 1.8 µm column (50 x 2.1 mm) fitted on a Waters system equipped with a Waters PDA detector (Baden-Dättwil, Switzerland). Buffer A = CH3CN ± 0.1 % formic acid), buffer B = H2O ± 0.1 % formic acid. Flow rate = 400.0 µL/min at 25 °C.

1H and ¹³C NMR spectra were recorded on Bruker Avance III Cryo 600 MHz spectrometers at 298 K. Chemical shifts (δ) are quoted in parts per million (ppm) and coupling constants (J) are in Hertz (Hz). s stands for singlet, d for doublet, dd for doublet of doublets, t for triplet, q for quartet, and m for multiplet. Residual solvent peaks were used as the internal reference for the proton and carbon chemical shifts.

NMR spectra were processed with Mnova version 8.1.2 software package.

Low resolution mass spectrometry (LRMS) was carried out on a HTS PAL-LC10A – API 150Ex instrument in ESI (positive mode). High resolution mass spectrometry (HRMS) was carried out on a Bruker Autoflex (MALDI-TOF) (reflectron mode).

Chemical structures were drawn and named according IUPAC nomenclature using ChemBioDraw Ultra version 14.0.0.117 software package.

Figures and statistical analysis were done using GraphPad Prism 7.02, (GraphPad Software Inc.) software. P-values < 0.05 were considered as statistically significant.

Gadolinium (Gd) quantification

The samples, blanks and calibration curves were diluted in a solution containing HNO3

2 %, butanol 1 % (VWR, Fontenay sous Bois, France) and Triton 0.5 % (Sigma, Buchs, Switzerland) in ultrapure water (resistivity greater than 18.2 MΩ cm). An external calibration curve in HNO3 2 % was prepared by monitoring the ¹⁵⁷Gd isotope with a dwell time of 300 ms in no-gas mode (means not using the collision cell). Internal standards (rhodium and indium; Agilent, Basel, Switzerland) were added at 10 μg/L to the analytical calibration solutions, analytical blanks and samples. The raw data were acquired using MassHunter software (Agilent, Tokyo, Japan).

Fluorescence binding assay of fibrin clots

First, fibrin clots were formed in a clear-bottom black 96-well plate (3603 Costar, USA).

In each well, 75 µL of a 2 mg/mL fibrinogen solution in 0.9 % NaCl and 30 µL of a 2.5 U/mL thrombin solution in 0.9 % NaCl were added. The plate was placed in a plate

4 shaker for 5 min at 37 °C and then incubated for 4 h at 37 °C. Second, mixes were prepared at different concentrations of DSPE-PEG2000-CREKA (3, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 mM), and mixes with DSPE-PEG2000-CERAK were prepared as a negative control. The fluorescent building block DSPE-PEG2000-IR-774 was also tested by itself. Third, 100 µL of the formulations were added on top of the fibrin clots and incubated for 1 h, and then fluorescence was measured for the first time before washing (ex 774 nm, em 806 nm (Synergy 2, Biotek™)). Fourth, the plates were washed 5 times with pure water, and the fluorescence was measured a second time. Finally, the results were normalized with regard to dye concentration in each formulation and plotted as normalized intensity as a function of CREKA or CERAK concentration (Fig. 6).

Nuclear magnetic resonance (NMR) relaxometry

Nuclear magnetic relaxation was measured on Bruker Minispec contrast agent analyzers mq20 (20 MHz, 0.47 T), mq30 (30 MHz, 0.71 T), mq40 (40 MHz, 0.93 T) and mq60 (60 MHz, 1.41 T) at 37.0 °C. Relaxation rates R1 (1/T1) were obtained by the inversion-recovery method and by the Carr-Purcell-Meiboom-Gill method.(Tear, Carrera et al. 2020) For all experiments, the spectrometers were equipped with Bruker BVT 3000 and Bruker BCU05 temperature control units.

5 Synthesis of Gd-DO3A-Tyr-(C18)2

6 Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 1

(tert-butoxycarbonyl)-L-tyrosine

Reaction:

Compound 1 was synthesized according to a published procedure.(Babic, Vorobiev et al. 2019)

In a 500 mL flask, 4.0 g of L-Tyrosine and 5.28 g of Boc2O are introduced with 100 mL of 1,4-dioxane, 50 mL H2O and 50 mL NaOH 1M. The mixture is left under magnetic stirring at room temperature for 3h, followed by an acidification to pH=2 with HCl 5M.

The mixture in extracted with 3x50 mL Ethyl Acetate. The organic phase is washed with 80 mL of brine, then dried on Na2SO4 and filtrated. Ethyl Acetate is evaporated under reduced pressure resulting in a 5.6 g of white solid and a yield of 90 %.

1H NMR (600 MHz, CDCl3): δ = 12.51 (s, 1H, COOH), 9.18 (s, 1H, NH), 7.01 (d, J = 6 Hz, 2H Ar), 6.64 (d, J = 6 Hz, 2H Ar), 3.99 (t, J = 6 Hz, 1H, CH), 2.86 (m, 1H, CH2), 2.70 (m, 1H, CH2), 1.32 (s, 9H, CH3)

13C NMR (151 MHz, CDCl3): δ = 173.73, 155.81, 155.44, 129.98, 128.01, 114.90, 77.98, 55.52, 35.66, 28.17

LRMS (ESI) m/z calculated for C14H19NO5 [M+H]⁺ 282.1 found 282.1

7

8 Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 2:

tert-butyl (S)-(1-(dioctadecylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate

Reaction:

Compound 2 was synthesized according to a published procedure.(Babic, Vorobiev et al. 2019)

Compound 1 (1.08 g, 3.83 mmol, 1 eq), diisopropylethylamine (1.27 mL (0.992 g), 7.66 mmol, 2 eq), HATU (1.53 g, 4.02 mmol, 1.05 eq), dioctadecylamine (2.00 g, 3.83 mmol, 1 eq) and chloroform (150 mL, c(Compound 1)=0.025 M) were stirred overnight at room temperature and under nitrogen atmosphere. The reaction mixture was filtered off and evaporated under reduced pressure. The crude product was purified by flash chromatography using hexane/ethyl acetate gradient to obtain a colorless solid (1.66 g, 2.11 mmol, 55 % yield).

1H NMR (600 MHz, CDCl3): δ = 7.02 (d, J = 6 Hz, 2H Ar), 6.68 (d, J = 6 Hz, 2H Ar), 5.72 (s, 1H, NH), 4.70 (s, 1H, CH), 3.42 (m, 2H, CH2), 3.03 (m, 2H, CH2), 2.94 (m, 2H, CH2), 1.41 (s, 9H, -CH3), 1.25 (m, 64H, aliphatic chains), 0.88 (t, J = 6 Hz, -CH3)

13C NMR (151 MHz, CDCl3): δ = 171.47, 155.21, 155.04, 130.78, 128.46, 115.40, 79.75, 51.76, 47.96, 46.58, 39.67, 32.08, 29.87, 29.52, 22.84, 14.27

LRMS (ESI) m/z calculated for C50H92N2O4 [M+H]⁺ 785.7 found 785.9

9

10

11 Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 3:

(S)-2-amino-3-(4-hydroxyphenyl)-N,N-dioctadecylpropanamide

Reaction:

1.5 g of Compound 2 was completely dissolved in 10 mL of DCM and 2 mL of TFA added to the solution. Reaction mixture was put under magnetic stirring for 1 h, and followed on TLC (solvent MeOH/DCM 9/1). Then the solvents were evaporated and the product washed 3 times with methanol and evaporated under reduced pressure to completely remove the TFA. Final yield is 98 %.

1H NMR (600 MHz, CDCl3): δ = 7.87 (s, 1H, OH), 7.00 (d, J = 6 Hz, 2H, Ar), 6.73 (d, J

= 6 Hz, 2H, Ar), 5.50 (s, 1H, NH), 4.43 (s, 1H, CH), 3.07 (m, 4H, CH2), 2.99 (m, 2H, CH2), 1.25 (s, aliphatic chains), 0.88 (t, J = 6 Hz, 6H, CH3)

13C NMR (151 MHz, CDCl3): δ = 167.53, 156.07, 130.78, 124.40, 114.71, 52.21, 48.01, 47.00, 36.82, 32.08, 29.87, 22.85, 14.27

LRMS (ESI) m/z calculated for C45H84N2O2 [M+H]⁺ 685.6 found 686.1

12

13

14 Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 4:

tri-tert-butyl 2,2',2''-(10-(2-((1-(dioctadecylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2- yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)(S)-triacetate

Reaction:

Amine (Compound 3, 0.196 mmol, 1 eq), DOTAtris(tBu)ester (0.294 mmol, 1.5 eq), N- (3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC, 0.588 mmol, 3 eq), 1-hydroxybenzotriazole hydrate (HOBt, 0.392 mmol, 2 eq), diisopropylethylamine (DIPEA, 0.600 mL, 6.26 mmol, 32 eq) were dissolved in dimethylformamide (DMF, 20 mL, c(compound 3)=0.01 M) and were stirred at room temperature under nitrogen atmosphere overnight at pH=8. After 24h the reaction mixture was evaporated under reduced pressure to obtain a yellow oil. The crude product was purified by flash chromatography three times (DCM/MeOH gradient) to obtain a yellow oily (wax) solid.

Final yield: 50 %.

1H NMR (600 MHz, CDCl3): δ = 7.82 (d, J = 36 Hz, 2H, Ar), 7.37 (s, 1H, OH), 6.89 (d, J = 18 Hz, 2H, Ar), 6.28 (s, 1H, NH), 5.04 (m, 1H, CH), 3.60 (m, 2H, CH2), 3.34 (m, 2H, CH2), 3.17 (m, 2H, CH2), 3.01 (m, 2H, CH2),2.83 (m, 2H, CH2),2.70 (m, 2H, CH2), 2.55 (m, 2H, CH2), 2.11 (m, 2H, CH2), 1.95 (m, 2H, CH2), 1.45 (s, 27H, CH3), 1.25 (s, aliphatic chains), 0.88 (t, J = 6 Hz, 6H, CH3)

13C NMR (151 MHz, CDCl3): δ = 172.73, 161.64, 157.43, 130.67, 130.28, 128.92, 116.52, 100.13, 55.87, 55.65, 46.52, 33.91, 32.07, 29.86, 29.51, 28.17, 28.04, 27.06, 25.06, 22.84, 14.27

LRMS (ESI) m/z calculated for C73H134N6O9 [M+H]⁺ 1240.0 found 1240.0, [M+Na]+ 1262.0 found 1262.3

15

16

17 Synthesis of Gd-DO3A-Tyr-(C18)2 – Compound 5:

(S)-2,2',2''-(10-(2-((1-(dioctadecylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2- yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

Reaction:

200 mg of compound 4 was dissolved in 2 mL of DCM and 2 mL of TFA added to the solution. Reaction mixture was put under magnetic stirring for 1 h, and followed on TLC (solvent MeOH/DCM 9/1). Then the solvents were evaporated and the product washed 3 times with methanol and evaporated under reduced pressure to completely remove the TFA. Final yield is 99 %.

1H NMR (600 MHz, CDCl3): δ = 7.05 (d, J = 6 Hz, 2H, Ar), 6.71 (d, J = 6 Hz, 2H, Ar), 3.88 (m, 2H, CH2), 3.06 (m, 2H, CH2), 2.94 (m, 2H, CH2), 2.85 (m, 2H, CH2), 1.29 (s, aliphatic chain), 0.90 (t, J = 6 Hz, 6H, CH3)

13C NMR (151 MHz, CDCl3): δ = 178.65, 171.37, 161.25, 161.01, 156.32, 130.30, 127.15, 127.00, 117.55, 115.62, 115.00, 37.83, 31.69, 29.40, 29.09, 28.46, 27.15, 26.64, 26.42, 26.21, 22.34, 13.05

LRMS (ESI) m/z calculated for C61H110N6O9 [M+H]⁺ 1071.8 found 1072.1

18

19 Synthesis of Gd-DO3A-Tyr-(C18)2 – Final step: Gadolinium loading

Reaction:

Was adapted from (Routledge, Jones et al. 2015)

To a solution of compound 5 in methanol, 1.05 eq of GdCl3 was added and the mixture was stirred at 40 °C for 30 min, then equal volume of ultrapure water was added, pH adjusted to 6 with NaOH 0.1 M and the reaction was left stirring at 40 °C for 48h. The reaction was followed by HPLC Thermo Fisher and the product purified on reverse phase HPLC Shimadzu (full gradient ACN/H2O, without TFA). Absence of free Gd was verified by xylenol orange method (Barge, Cravotto et al 2006). White powder was obtained. Final yield 63%.

1H NMR (600 MHz, CDCl3): deformed due to Gd

13C NMR (151 MHz, CDCl3): deformed due to Gd

HRMS (MALDI) m/z calculated for C61H107GdN6O9 [M+H]⁺ 1226.74 found 1226.88

20

21 HPLC spectrum of Gd-DO3A-Tyr-(C18)2 after purification

22 Synthesis of DSPE-PEG2000-CREKA (and CERAK)

Reaction:

Adapted from (Yoo, Pineda et al. 2016).

CREKA (10 mg - 0.016 mmol) and DSPE-PEG2000-Maleimide (60 mg – 0.02 mmol) were dissolved in DMF (5 mL), then DIPEA (200 mg) was added. The reaction was stirred at room temperature for 24 h under N2. The mixture was evaporate and dissolved in MeOH. Finally the solution was purified on HPLC Shimadzu (full gradient ACN/H2O). A white powder was obtained. Final yield 82 %.

HRMS (MALDI) m/z calculated for C162H314N13O65PS [M+H]⁺ 3545.11 found see below

23 Superposition: DSPE-PEG2000-Mal and DSPE-PEG2000-CREKA

Superposition: DSPE-PEG2000-Mal and DSPE-PEG2000-CERAK

24 Synthesis of DSPE-PEG2000-IR-774 – all steps

25 Synthesis of DSPE-PEG2000-IR-774 – Step 1: Compound 1

2-((E)-2-((E)-2-chloro-3-(2-((E)-1-(4-ethoxy-4-oxobutyl)-3,3-dimethylindolin-2-

ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(4-ethoxy-4-oxobutyl)-3,3-dimethyl-3H- indol-1-ium

Reaction:

1. To a solution of 14 ml POCl3 in 7.5 ml dichloromethane (extra dry, stabilized, on molecular sieves, 34846, AcrosSeal), 15 ml of N,N-diméthylformamide (extradry, on molecular sieves, 40248, Sigma Aldrich) and 7,5 ml of dichloromethane (extra dry, stabilized, on molecular sieves, 34846, AcrosSeal) were added under nitrogen atmosphere at 0 °C. To this mixture was added 3.96 mL of cyclohexanone dropwise at 0 °C. The mixture was heated at 60 °C for 3 hours, under stirring. After the reaction was allowed to cool down, 75 g of ice water was added. The organic phase was discarded and the aqueous phase was filtered out to obtain a yellow precipitate further dried under vacuum. (m=1.76 g, yield=26%)

2. To a solution of 133 mg of “bisaldehyde” (see 1 above) in 12 mL of acetic anhydride, was added 42 mg of ethyl-4-bromobutyrate. To the mixture was then added 127 mg of anhydrous sodium acetate. The reaction was carried under nitrogen atmosphere at 80

°C for 1.5 hour under stirring.

An aqueous solution of 20 % NaBr was added to the mixture to precipitate the product.

The resulting mixture was kept a 4 °C overnight and precipitated by filtration to obtain a green product further dried under vacuum. The product was then purified by flash chromatography with a gradient of DCM/MeOH 9/1 (m=226 mg, yield=43%).

1H NMR (600 MHz, CDCl3): δ = 8.32 (d, J = 6 Hz, 2H, Ar), 7.39 (dd, J = 6 Hz, 2H, Ar), 7.35 (q, J = 6 Hz, 4H, Ar), 7.24 (q, J = 6 Hz, 2H, CH), 6.51 (q, J = 6 Hz, 2H, CH), 4.38 (t, J = 6 Hz, 4H, CH2), 4.15 (q, J = 6 Hz, 4H, CH2), 2.83 (t, J = 6 Hz, 4H, CH2), 2.66 (t, J = 6 Hz, 4H, CH2), 2.13 (m, 4H, CH2), 1.99 (m, 2H, CH2), 1.72 (s, 12H, CH3), 1.27 (t, J = 6 Hz, 6H, CH3)

13C NMR (151 MHz, CDCl3): δ = 173.18, 172.24, 150.11, 144.34, 142.44, 141.18, 129.08, 128.61, 125.30, 122.23, 111.29, 102.28, 60.93, 49.37, 44.09, 30.81, 28.31, 27.08, 22.43, 20.99, 14.37

HRMS (MALDI) m/z calculated for C42H52ClN2O4 [M+H]⁺ 684.36 found 684.34

26

27

28 Synthesis of DSPE-PEG2000-IR-774 – Step 2: final structure

Reaction:

Adapted from (Noh, Lee et al. 2018)

In a 2 mL Biotage® tube, we added 25 mg of DSPE-PEG2000-amine dissolved in 750 µL of dry DMF. To this mixture, we added 10 mg of compound 1 (1.5 eq) and 15 µL of triethylamine dissolved in 750 µL of dry DMF. The reaction was put in the microwave oven at 140 °C for 45 min, Absorption: very high, pre-stirring: 30 seconds. After that, we evaporated the solvent and stored the product at -20 °C. We obtained a dark blue product. Final yield: 86%. The product was analyzed by MALDI-TOF.

HRMS (MALDI) m/z calculated for C174H314N4O58P [M+H]⁺ 3420.15 found see below:

29 Absorbance and emission spectrums of DSPE-PEG2000-IR-774

Methods:

Concentration of the dye in solution was fixed to 0.005 mM in Mili-Q water for these experiments. Wavelength of maximum absorbance were determined on UV-1800 Spectrophotometer (Shimadzu, Tokyo, Japan) and maximum fluorescence emission on Fluoromax-4 spectrofluorometer (HORIBA Scientific, Kyoto, Japan).

Maximum absorbance: 774 nm Epsilon value: ε = 264’000 [mol/dm³]

Figure SI1. UV-Visible spectrum of DSPE-PEG2000-IR-774 in water solution 0.005 mM

Maximum fluorescence (excitation at ex=774nm): 806 nm

Figure SI2. Corrected emission spectrum of DSPE-PEG2000-IR-774 in water solution 0.05 mM, at room temperature, upon excitation at ex=774 nm

30 Cell internalization and fluorescence imaging: cells selection images of Mixes- 1,2,3 and Anti-Mixes-1,2,3 – MCF-7 cell line

Figure SI3. Cell internalization assay. Fluorescence imaging on three different channels on MCF-7 cell lines for the three mixes and anti-mixes.

31 Cell internalization and fluorescence imaging: cells selection images of Mixes- 1,2,3 and Anti-Mixes-1,2,3 – MCF-10 cell line

Figure SI4. Cell internalization assay. Fluorescence imaging on three different channels on MCF-10 cell lines for the three mixes and anti-mixes

32 Cell internalization and fluorescence imaging: fluorescence quantification and graphical presentation

0 2 0 4 0 6 0 8 0 1 0 0

0 21 0⁶ 41 0⁶ 61 0⁶ 81 0⁶

D S P E -P E G_{2 0 0 0}- I R - 7 7 4 ( f lu o r e s c e n t d y e ) C o n c e n tr a tio n

[µ M ] Fluorescence Integrated Intensity Sum

C o n tr o l s M C F - 1 0 M ix 1 M C F - 1 0 M ix 2 M C F - 1 0 M ix 3 M C F - 1 0

A n ti-M ix 1 M C F - 1 0 A n ti-M ix 2 M C F - 1 0 A n ti-M ix 3 M C F - 1 0

C o n tr o l s M C F - 7 M ix 1 M C F - 7 M ix 2 M C F - 7 M ix 3 M C F - 7

A n ti-M ix 1 M C F - 7 A n ti-M ix 2 M C F - 7 A n ti-M ix 3 M C F - 7

Figure SI5. Cell internalization assay. Fluorescence quantification in the whole well (fluorescence integrated intensity sum) in function of the dye concentration.

33 References:

Babic, A., V. Vorobiev, G. Trefalt, L. A. Crowe, L. Helm, J. P. Vallee and E. Allemann (2019). "MRI micelles self-assembled from synthetic gadolinium-based nano building blocks." Chem Commun (Camb) 55(7):

945-948.

Noh, I., D. Lee, H. Kim, C. U. Jeong, Y. Lee, J. O. Ahn, H. Hyun, J. H. Park and Y. C. Kim (2018). "Enhanced Photodynamic Cancer Treatment by Mitochondria-Targeting and Brominated Near-Infrared Fluorophores." Advanced Science 5(3).

Routledge, J. D., M. W. Jones, S. Faulkner and M. Tropiano (2015). "Kinetically Stable Lanthanide Complexes Displaying Exceptionally High Quantum Yields upon Long-Wavelength Excitation: Synthesis, Photophysical Properties, and Solution Speciation." Inorganic Chemistry 54(7): 3337-3345.

Tear, L. R., C. Carrera, E. Gianolio and S. Aime (2020). "Towards an Improved Design of MRI Contrast Agents: Synthesis and Relaxometric Characterisation of Gd-HPDO3A Analogues." Chemistry.

Yoo, S. P., F. Pineda, J. C. Barrett, C. Poon, M. Tirrell and E. J. Chung (2016). "Gadolinium-Functionalized Peptide Amphiphile Micelles for Multimodal Imaging of Atherosclerotic Lesions." Acs Omega 1(5): 996- 1003.