S1

Supporting Information for:

Multisensitive drug-loaded polyurethane/polyurea

nanocapsules with pH-synchronized shell cationization

and redox-triggered release

Cristina Cuscó,*1,2 Jordi Garcia,2 Ernesto Nicolás,2 Pau Rocas1 and Josep Rocas*1

1 Nanobiotechnological Polymers Division, Ecopol Tech, S.L., El Foix Business Park, Indústria 7,

43720 L’Arboç del Penedès, Tarragona, Spain

2 Organic Chemistry Section, Inorganic and Organic Chemistry Department, Faculty of

Chemistry, CIBERobn and IBUB, University of Barcelona, Martí i Franquès 1-11, 08028

Barcelona, Spain

1. Acronyms

IPDI: isophorone diisocyanate. It is an organic aliphatic diisocyanate. Interestingly, aliphatic

isocyanates are less pulmonary sensitizing and not carcinogenic compared to the aromatic

ones. It is particularly noteworthy that fully reacted isocyanate polymers to form their

respective urethane or urea bonds do not contain toxicity referred to the isocyanate group.[1]

In addition, degradation products of IPDI, such as isophorone diamine are rapidly eliminated

via urinary excretion.[2]

DEDS: 2-hydroxyethyl disulfide.

YMER N-120: polymeric non-ionic hydrophilic building block containing two primary hydroxyl

groups and a long capped ethoyxylated side chain. From a synthetic point of view, YMER N-120

was selected because of its low melting point and viscosity.

Genamin TAP 100D: N-tallow-1,3-propylenediamine. It is a hydrophobic building block

containing C18 fatty chain and two reactive amines for polymerization.

Jeffcat DPA: N-(3-dimethylaminopropyl)-N,N-diisopropanolamine. It contains two cationizable

tertiary amines and two alcohols for polymerization.

THF: tetrahydrofurane.

Electronic Supplementary Material (ESI) for Polymer Chemistry.This journal is © The Royal Society of Chemistry 2016

S2

DETA: diethylenetriamine. It is a water-soluble molecule used as a crosslinker and it contains

two primary amines and one secondary amine.

HCl: hydrochloric acid.

DiO: 3,3’-dioctadecyloxacarbocyanine perchlorate (lipophilic tracer).

DiI: 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (lipophilic tracer).

MWCO: molecular weight cut off.

FT-IR (ATR): Fourier transform infrared spectroscopy (attenuated total reflectance).

DLS: dynamic light scattering.

TEM: transmission electron microscopy.

AFM: atomic force microscopy.

UV-Vis: ultraviolet-visible spectroscopy.

DL: drug loading.

EE: encapsulation efficiency.

FRET: Förster resonance energy transfer.

PBS: phosphate buffered saline.

HSA: human serum albumin.

BSA: bovine serum albumin.

L-GSH: reduced L-glutathione.

HLB: hydrophilic lipophilic balance.

EPR: enhanced permeability and retention.

S3

2. Figures

Composition (% by weight) of each polymer

Synthesis of the amphiphilic cationic prepolymer (P1)

Monomer % by weight

DEDS 3.524

Ymer N-120 40.820

Jeffcat DPA 3.432

IPDI 29.322

TAP 100 D 22.903

Synthesis of the amphiphilic prepolymer (P2)

Monomer % by weight

DEDS 4.098

Ymer N-120 47.467

Jeffcat DPA 0

IPDI 27.149

TAP 100 D 21.286

Synthesis of non-labile amphiphilic and amphoteric prepolymer (P3)

Monomer % by weight

1,6-hexanediol 2.705

Ymer N-120 41.166

Jeffcat DPA 3.461

IPDI 29.571

TAP 100 D 23.097

Figure S1. Composition of each polymer in % by weight.

S4

Nanoencapsulation process

(A)

(B)

(C)

S5

(D)

S6

(E)

Figure S2. Main species present in each step. (A) The monomers are loaded; (B) the

amphiphilic prepolymer is formed; (C) the desired hydrophobic drug (purple dots) is added into

the polymeric mixture; (D) an aminic ionomer is introduced and cold water is added and the

nanoemulsion is formed; (E) a polyamine (yellow dots) is added to crosslink the nanocapsules.

S7

Figure S3. FT-IR spectrum corresponding to steps 1a, 1b and 2 of the synthetic process

(amphiphilic prepolymer P1 preparation).

Figure S4. FT-IR spectrum corresponding to steps 2, 3 and 4 of the nanoencapsulation process

(amphiphilic prepolymer P1 preparation).

Step 1a

Step 1b

Step 2

Step 2

Step 3

Step 4

S8

Figure S5. Measurement of pH over time after crosslinker addition.

Figure S6. TEM micrographs of nanocapsules loaded with: (A) paclitaxel, (B) curcumin, (C) HL3

and (D) HL4.

7.5

8

8.5

9

9.5

10

10.5

11

11.5

0 100 200 300

pH

Time (min)

Amine consumption

A B

C D

S9

Figure S7. AFM micrographs of nanocapsules loaded with paclitaxel in: (A) 2D and (B) 3D; scale

bar in nanometres.

Figure S8. Z-potential measurements at different pH conditions of amphiphilic anionic

nanocapsules (P2-drug loaded anionic NCs) and surface charge at: (A) extracellular

microenvironmental pH and (B) blood pH.

-20

-10

0

10

5 5.5 6 6.5 7 7.5 8 8.5

Z-p

ote

nti

al (

mV

)

pH

Z-potential vs. pH (anionic NCs)

A B

B A

A B

S10

Figure S9. Z-potential measurements at different pH conditions of amphiphilic cationic

nanocapsules (P1-drug-loaded cationic NCs) and surface charge at: (A) microenvironmental

extracellular pH and (B) blood pH.

Figure S10. Formulae to calculate %EE and %DL of the nanocapsules.

0

10

20

30

40

50

60

5 5.5 6 6.5 7 7.5 8 8.5

Z-p

ote

nti

al (

mV

)

pH

Z-potential vs. pH (cationic NCs)

A B

B A

S11

Figure S11. Size distribution by number of nanocapsules under control conditions (PBS),

measured at different periods of time.

Fluorescence studies

Figure S12. Fluorescence studies with DiO- and DiI-loaded NCs in control conditions (PBS).

0

5

10

15

20

25

30

35

1 10 100 1000 10000

Nu

mb

er

(%)

Size (nm)

Size distribution by number

0 h

24 h

48 h

72 h

96 h

0 E+00

1 E+04

2 E+04

3 E+04

4 E+04

5 E+04

6 E+04

7 E+04

490 510 530 550 570 590 610 630 650

Inte

nsi

ty

Wavelength (nm)

Assay with PBS PBS 0h PBS 0.5h

PBS 1h PBS 1.5h

PBS 2h PBS 3h

PBS 4h PBS 5h

PBS 6h PBS 7h

PBS 8h PBS 9h

PBS 10h PBS 24h

PBS 48h PBS 72h

PBS 96h

S12

Figure S13. Fluorescence studies with DiO- and DiI-loaded NCs in HSA.

Figure S14. Fluorescence studies with DiO- and DiI-loaded NCs in BSA.

0 E+00

2 E+04

4 E+04

6 E+04

8 E+04

1 E+05

1 E+05

490 510 530 550 570 590 610 630 650

Inte

nsi

ty

Wavelength (nm)

Assay with HSA HSA 0h HSA 0.5h

HSA 1h HSA 1.5h

HSA 2h HSA 3h

HSA 4h HSA 5h

HSA 6h HSA 7h

HSA 8h HSA 9h

HSA 10h HSA 24h

HSA 48h HSA 72h

HSA 96h

0 E+00

2 E+04

4 E+04

6 E+04

8 E+04

1 E+05

1 E+05

490 510 530 550 570 590 610 630 650

Inte

nsi

ty

Wavelength (nm)

Assay with BSA BSA 0h BSA 0.5h

BSA 1h BSA 1.5h

BSA 2h BSA 3h

BSA 4h BSA 5h

BSA 6h BSA 7h

BSA 8h BSA 9h

BSA 10h BSA 24h

BSA 48h BSA 72h

BSA 96h

S13

Figure S15. Fluorescence studies with DiO- and DiI-loaded NCs in reductive conditions (L-GSH

10 mM).

0 E+00

1 E+04

2 E+04

3 E+04

4 E+04

5 E+04

6 E+04

7 E+04

8 E+04

9 E+04

490 510 530 550 570 590 610 630 650

Inte

nsi

ty

Wavelength (nm)

Assay with L-GSH PBS 0h PBS 0.5hPBS 1h PBS 1.5hGSH 0h GSH 1hGSH 2h GSH 3hGSH 4h GSH 5hGSH 6h GSH 7hGSH 8h GSH 22hGSH 24h GSH 25hGSH 26h GSH 27hGSH 28h GSH 29hGSH 30h GSH 48hGSH 49h GSH 50hGSH 51h GSH 52hGSH 53h GSH 54hGSH 70h GSH 71hGSH 94h

S14

Figure S16. Slope calculated from FRET ratio over time with DiO- and DiI-loaded NCs in

reductive conditions (L-GSH 10 mM)

y = 0.0038x + 0.2179

0.22

0.23

0.24

0.25

0.26

0 2 4 6 8 10 12

FRET

rat

io

Time (h)

GSH (I)

y = 0.0125x + 0.1329

0

0.2

0.4

0.6

0.8

1

20 30 40 50 60

FRET

rat

io

Time (h)

GSH (II)

y = 0.0010x + 0.8066

0.5

0.6

0.7

0.8

0.9

1

70 75 80 85 90 95 100

FRET

rat

io

Time (h)

GSH (III)

S15

Figure S17. Slope calculated from FRET ratio over time with DiO- and DiI-loaded NCs under

control conditions (PBS).

Figure S18. Slope calculated from FRET ratio over time with DiO- and DiI-loaded NCs in HSA.

Figure S19. Slope calculated from FRET ratio over time with DiO- and DiI-loaded NCs in BSA.

y = 0.0003x + 0.2325

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

FRET

rat

io

Time (h)

PBS

y = 0.0003x + 0.2300

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

FRET

rat

io

Time (h)

BSA

y = 0.0002x + 0.2483

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80 100

FRET

rat

io

Time (h)

HSA

S16

Figure S20. Fluorescence studies with non-labile DiO- and DiI-loaded NCs under reductive

conditions (L-GSH 10 mM).

Figure S21. Slope calculated from FRET ratio over time with non-labile DiO- and DiI-loaded NCs

under reductive conditions (L-GSH 10 mM).

0 E+00

2 E+04

4 E+04

6 E+04

8 E+04

1 E+05

1 E+05

490 510 530 550 570 590 610 630 650

Inte

nsi

ty

Wavelength (nm)

Assay with GSH (non-labile NCs) PBS 0h PBS 0.5h

PBS 1h PBS 1.5h

GSH 0h GSH 1h

GSH 2h GSH 3h

GSH 4h GSH 5h

GSH 6h GSH 7h

GSH 20h GSH 21h

GSH 22h GSH 23h

GSH 24h GSH 25h

GSH 48h GSH 55h

GSH 68h GSB 72h

GSH 94h

y = 0.0003x + 0.2610

0

0.2

0.4

0.6

0.8

1

0 20 40 60 80

FRET

rat

io

Time (h)

GSH (I) (non-labile NCs)

y = 0.0032x + 0.0399

0

0.2

0.4

0.6

0.8

1

70 75 80 85 90 95 100

FRET

rat

io

Time (h)

GSH (II) (non-labile NCs)

S17

3. References

[1] US Environmental Protection Agency_Diisocyanates Toxicology.

http://www.epa.gov/oppt/auto/profile/toxicology1a.pdf (accessed May 20, 2016).

[2] L.T. Budnik, D. Nowak, R. Merget, C. Lemiere and X. Baur, J. Occup. Med. Toxicol., 2011, 6,

9.