S1

SUPPLEMENTARY INFORMATION

Formulation of benzoxaborole drugs in PLLA:

from materials preparation to in vitro release kinetics and cellular assays

S. Sene, J. McLane, N. Schaub, S. Bégu, P. H. Mutin, L. Ligon, R. J. Gilbert, D. Laurencin

Content Page

Figure S1. TGA curves of a) neat PLLA, PLLA-0 and PLLA-10%AN2690 and b) PLLA-0, PLLA-0-vacuum (dried under vacuum) and PLLA-0-H (heated at 60 °C).

S2

Figure S2. 1H solution NMR spectra of PLLA-0 and PLLA-10%AN2690. S2

Table S1. Amount of residual chloroform in films with /without post-treatments. S3

Figure S3. a) DSC curves of PLLA-0 and PLLA-0-H and b) Crystallinity of PLLA-0, PLLA-3%AN2690 and PLLA-10%AN2690 before and after the heat treatment, as determined by DSC.

S3

Figure S4: X-ray diffractograms of PLLA-0 and PLLA-10%AN2690, before and after the heat-treatment, in comparison with crystalline AN2690.

S4

Figure S5: a) 11

B solid state NMR spectra of PLLA films loaded with AN2690 at different wt %, before and after the heat-treatment ; b)

13C{

19F} REDOR NMR study of PLLA-10%AN2690 before and after the heat-treatment.

S4

Figure S6: a) Water contact angles of film samples with PLLA-PEO loaded with different wt % of AN2690, compared to PLLA-0; b)

11B solid state NMR spectra of PLLA-PEO films loaded with AN2690 at different wt %; c)

19F solid state NMR spectrum of PLLA-PEO-10%AN2690 and its simulation considering two sites.

S5

Figure S7: a) TEM images of PLLA-10%LDH-0 and PLLA-10%LDH-AN2690; b) X-ray diffractograms of PLLA-0, LDH-AN2690 and PLLA-10%LDH-AN2690; c)

11B solid state NMR spectra of AN2690, LDH-AN2690, PLLA-

10%LDH-AN2690.

S6

Table S2: Fitting parameters for the drug-release curves of PLLA-x%AN2690 formulations, using zero-order, first order and Fickian-diffusional (Higuchi-like) models, in the “burst” and “sustained” release regions.

S7

Table S3: Fitting parameters for the drug-release curves of different film formulations, using a Fickian-diffusional model (Higuchi-like), in the sustained-release region.

S7

Figure S8: Release kinetics of PLLA film formulations, for samples at 10 and 25 wt% AN2690. a) PLLA-10%AN2690-H and PLLA-25%AN2690-H, in comparison with the non-heated samples. b) PLLA-PEO-10%AN2690 and PLLA-PEO-25%AN2690, in comparison with the samples without PEO

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Figure S9: a) Average molecular weight (Mn) and b) crystallinities (Xc) of PLLA-0, PLLA 3%AN2690 and PLLA-10%AN2690 (heated and non-heated), before and after exposure to PBS at 37 °C for 20 days.

S9

Figure S10: 11

B solid state NMR spectra of PLLA-10%LDH-BBzx before and after 5 days of release in PBS. S10

Figure S11: Image of the migration assay on MDA-MB-231 cells at day-5 (molecules in solution). S10

Figure S12: Morphology assays on MDA-MB-231 cancer cells, showing the evolution of normalized cell areas, according to the nature of the organoboron molecules in solution.

S11

Figure S13: Study on the effect of organoboron molecules on MDA-MB-231 cancer cell migration (day 5): release from heat-treated PLLA-based film formulations with different drug contents.

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Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2015

S2

Figure S1:TGA curves of a) neat PLLA, PLLA-0 and PLLA-10%AN2690 and b) PLLA-0, PLLA-0-

vacuum (dried under vacuum) and PLLA-0-H (heated at 60 °C).

Figure S2: 1H solution NMR spectra of PLLA-0 and PLLA-10%AN2690.

When PLLA-0 was dissolved in CDCl3, no CH2Cl2 signal was observed, while when PLLA was dissolved

in CD2Cl2, the CHCl3 signal was observed. This means that the residual solvent in the films

corresponds to chloroform. The chloroform signal is also present for PLLA-10%AN2690, but in a much

smaller quantity.

80

85

90

95

100

25 125 225 325 425

TG / (%)

Temperature (°C)

PLLA-0

PLLA-0-vacuum

PLLA-0-H0

20

40

60

80

100

25 125 225 325 425

TG / (%)

Temperature (°C)

Neat PLLA

PLLA-0

PLLA-10%AN2690

a/ b/

1H chemical shift (ppm)

45678

PLLA-0 in CD2Cl2

PLLA-0 in CDCl3

PLLA-10%AN2690 in CD2Cl2

chloroform

chloroform

S3

Table S1: Amount of residual chloroform in films with /without post-treatments.

The estimation was obtained with 1H solution NMR, by calculating the ratio between the intensities

of the chloroform peak at 7.32 ppm and the proton of the CH group of PLLA at 5.17 ppm

(quadruplet).

Figure S3: a) DSC curves of PLLA-0 and PLLA-0-H and b) Crystallinity of PLLA-0, PLLA-3%AN2690 and

PLLA-10%AN2690 before and after the heat treatment, as determined by DSC (n = 3; **p<0.05).

Post-treatmentPLLA-0 PLLA-10%BBzx

wt% wt%

- 12.8 8.1

Vacuum 11.3 5.7

Heat treatment (60 °C) 6.0 1.4

0

20

40

60

Xc

(%)

Non-heated

Heated

** **

20

22

24

26

28

30

0 50 100 150 200

Hea

t Fl

ow

(m

W)

Temperature (°C)

PLLA-0

PLLA-0-H

b/a/

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Figure S4: X-ray diffraction diagrams of PLLA-0 and PLLA-10%AN2690, before and after the heat-

treatment, in comparison with crystalline AN2690.

Figure S5: a) 11B solid state NMR spectra of PLLA films loaded with AN2690 at different wt %,

before and after the heat-treatment; b) 13C{19F} REDOR NMR study of PLLA-10%AN2690 before and

after the heat-treatment: spectra recorded with (red) and without (black) 19F recoupling pulses

were compared.

X

XRD

2θ ( )10 20 30 40 50 60 70

AN2690

PLLA-10%AN2690-H

PLLA-0

PLLA-0-H

PLLA-10%AN2690

a/

11B chemical shift (ppm)

-5051015202530354045

PLLA-25%AN2690

PLLA-10%AN2690

PLLA-3%AN2690

Non-heatedHeated

13C chemical shift (ppm)

020406080100120140160180

S0

S

12

3

PLLA-10%AN2690

PLLA-10%AN2690-H

b/

S5

Figure S6: a) Water contact angles of film samples with PLLA-PEO loaded with different wt % of

AN2690, compared to PLLA-0 (n = 6; *p<0.005); b) 11B solid state NMR spectra of PLLA-PEO films

loaded with AN2690 at different wt %; c) 19F solid state NMR spectrum of PLLA-PEO-10%AN2690

(blue) and simulated spectrum (red) considering two sites (green and purple).

11B chemical shift (ppm)

-10-505101520253035404550

25%AN2690

10%AN2690

3%AN2690

PLLA-PEOPLLA

0

20

40

60

80C

on

tact

an

gle

(°)

a/

b/

c/

19F chemical shift (ppm)

-115-110-105

PLLA-PEO-10%AN2690Exp

PLLA-PEO-10%AN2690Simul

*

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Figure S7: a) TEM images of PLLA-10%LDH-0 and PLLA-10%LDH-AN2690; b) X-ray diffractograms

of PLLA-0, LDH-AN2690 and PLLA-10%LDH-AN2690; c) 11B solid state NMR spectra of AN2690,

LDH-AN2690, PLLA-10%LDH-AN2690.

b/ XRD

11B chemical shift (ppm)

010203040

PLLA-10% LDH-BBzx

LDH-AN2690

AN2690

2θ ( )

10 20 30 40 50 60

LDH-AN2690

PLLA-0

PLLA-10%LDH-AN2690

PLLA-10%LDH-AN2690

2 µm5 µm 200 nm

PLLA-10%LDH-0

5 µm 2 µm 200 nm

a/ TEM

c/ 11B MAS solid state NMR

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Table S2. Fitting parameters for the drug-release curves of PLLA-x%AN2690 formulations (x = 3, 10,

25), using zero-order, first order and Fickian-diffusional (Higuchi-like)1 models, in the “burst” and

“sustained” release regions.

Table S3. Fitting parameters for the drug-release curves, using a Fickian-diffusional model (Higuchi-

like),1 in the sustained-release region. The following equation was used for the fits:

Mt/M∞ = kt0.5 + b

Sample time range (days)

Equation parameters R2

type x k b

PLLA-x%AN2690 3 1-30 0.0125 0.0575 0.993

10 1-30 0.0206 0.1268 0.994

PLLA-x%AN2690-H 3 1-15 0.0435 0.0741 0.994

10 1-15 0.0482 0.0561 0.995

PLLA-PEO-x%AN2690 3 1-15 0.0081 0.2572 0.998

10 1-15 0.0134 0.2741 0.988

PLLA-LDH-AN2690 - 1-15 0.0270 0.4441 0.880

1 J. Siepmann, N. A. Peppas, Int. J. Pharm. 2011, 418, 6.

Model SampleTime range

(days)k b R2

Zero-orderMt/M∞ = kt + b

PLLA-3%AN2690

0 – 0.25 0.2873 0 0.808

1 – 30 0.0020 0.0742 0.940

PLLA-10%AN2690

0 – 0.25 0.6131 0 -1,797

1 – 30 0.0032 0.1544 0.927

PLLA-25%AN2690

0 – 0.25 2.309 0 -0.044

1 – 30 0.0017 0.5508 0.769

First-orderln(1- Mt/M∞) = kt + b

PLLA-3%AN2690

0 – 0.25 -0.2942 0 0.819

1 – 30 -0.0022 -0.0769 0.943

PLLA-10%AN2690

0 – 0.25 -0.6434 0 -1.458

1 – 30 -0.0040 -0.1673 0.935

PLLA-25%AN2690

0 – 0.25 -2.8545 0 0.423

1 – 30 -0.0040 -0.8003 0.785

Fickian-diffusionalMt/M∞ = kt0.5 + b

PLLA-3%AN2690

0 – 0.25 0.1108 0 0.848

1 – 30 0.0125 0.0575 0.993

PLLA-10%AN2690

0 – 0.25 0.2466 0 0.743

1 – 30 0.0206 0.1268 0.994

PLLA-25%AN2690

0 – 0.25 0.9175 0 0.944

1 – 30 0.0117 0.5343 0.900

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Figure S8: Release kinetics of PLLA film formulations, for samples at 10 and 25 wt% AN2690. a)

PLLA-10%AN2690-H and PLLA-25%AN2690-H, in comparison with the non-heated samples. b) PLLA-

PEO-10%AN2690 and PLLA-PEO-25%AN2690, in comparison with the samples without PEO. Data

are mean +/- standard deviation (n=4 except for PLLA-10%AN2690-H where n=3).

0

20

40

60

80

0 2 4 6 8 10 12 14

Cu

mu

lati

ve r

ele

ase

(%)

time (days)

PLLA-25%AN2690

PLLA-25%AN2690-H

0

10

20

30

40

50

60

70

0 2 4 6 8 10 12 14

Cu

mu

lati

ve r

ele

ase

(%)

time (days)

PLLA-25%AN2690

PLLA-PEO-25%AN2690

a/ Heat-treated PLLA b/ PLLA-PEO blends

0

5

10

15

20

25

0 2 4 6 8 10 12 14

Cu

mu

lati

ve r

ele

ase

(%)

time (days)

PLLA-10%AN2690

PLLA-10%AN2690-H

0

10

20

30

40

0 2 4 6 8 10 12 14

Cu

mu

lati

ve r

ele

ase

(%)

Time (days)

PLLA-10%AN2690

PLLA-PEO-10%AN2690

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Figure S9: a) Average molecular weight (Mn) and b) crystallinities (Xc) of PLLA-0, PLLA-3%AN2690

and PLLA-10%AN2690 (heated and non-heated), before and after exposure to PBS at 37 °C for 20

days. Data are mean +/- standard deviation (n=3); *p<0.005, **p<0.05.

Non-heated Heated

a/ Average molecular weight

b/ Crystallinity

Non-heated Heated

0

20

40

60

80

Mn

(kD

a)

t = 0 day

t = 20 days

******

0

20

40

60

80

Mn

(kD

a)

t = 0 day

t = 20 days

*** **

0

20

40

60

Xc

(%)

t = 0 day

t = 20 days

** **

0

20

40

60

Xc

(%) t = 0 day

t = 20 days

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Figure S10: 11B solid state NMR spectra of PLLA-10%LDH-BBzx before and after 5 days of release in

PBS.

Figure S11: Image of the migration assay on MDA-MB-231 cells at day-5 (molecules in solution). As

detailed in the experimental section, cells were stained with Coomassie brilliant blue, before

imaging the plate and then drawing a circle around the cell mass using imageJ.

11B chemical shift (ppm)

-10-5051015202530354045

PLLA-10%LDH-BBzx

t ~ 5 days

t = 0

1.0 mM 0.5 mM 0.1 mM

CTRL

PBA

AN2690

BBzx

S11

Figure S12: Morphology assays on MDA-MB-231 cancer cells, showing the evolution of normalized

cell areas, according to the nature of the organoboron molecules in solution (1.0 mM

concentration). For the control experiment (CTRL), no organoboron molecule was present in the

medium. Data are mean +/- standard deviation (n=3).

Figure S13: Study on the effect of organoboron molecules on MDA-MB-231 cancer cell migration

(day 5): release from heat-treated PLLA-based film formulations with different drug contents.

Comparison of the quantity of drug released based on release kinetic assays (left) and the

corresponding normalized coverage area (right). For the control experiments (CTRL), no

benzoxaborole molecules were present in the film. Data are mean +/- standard deviation (n=3);

*p<0.005, **p<0.05.

AN2690

BBzx

PBA

0%

20%

40%

60%

80%

100%

d0 d2 d5

No

rmal

ize

d c

ell

are

a

CTRL

PBA

BBzx

AN2690

time (days)

a/ b/

0

5

10

15

20

25

30

No

rmal

ize

d r

ele

ase

(w

t%)

0%

20%

40%

60%

80%

100%

No

rmal

ize

d C

ove

rage

Are

a

* ** **