S1

Supporting Information

A 3D Printed Drug Delivery Implant formed from a Dynamic Supramolecular

Polyurethane Formulation

S. Salimi,a, Y. Wu,b, M. I. Evangelista Barreiros,b A. A. Natfji,c S. Khaled,d R. Wildman,b L.R. Hart,a F. Greco,c E. A. Clark,d C. J. Roberts*d and W. Hayes*a

a Department of Chemistry, University of Reading, Whiteknights, Reading, RG6 6AD, UK email: w.c.hayes@reading.ac.uk. b Faculty of Engineering, The University of Nottingham, University Park, Nottingham, NG7 2RD, UK c School of Pharmacy, University of Reading, Whiteknights, Reading, RG6 6AD, UK d School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK

Synthesis of N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine 1…………………………….S2Figure S1: 1H NMR spectrum of N-(4-methoxybenzyl)-1-(4-nitrophenyl)methanamine....................S3Figure S2: 13C NMR of N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine. .................................S3Synthesis of SPU1……………………………………………………...……………………………..S4Figure S3: 1H NMR spectrum of the polyurethane SPU1. ...................................................................S4Figure S4: 13C NMR spectrum of the polyurethane SPU1. ..................................................................S5Figure S5: Stacked 1H NMR spectra of the end-group, Krasol HLBH-P2000 2, and SPU1 ...............S5Figure S6: Infrared spectrum of SPU1. ................................................................................................S6Figure S7: GPC eluograms of SPU1 and Krasol HLBH-P2000. .........................................................S6Figure S8: Differential Scanning Calorimetry of SPU1, F1 and F2 cast and printed..........................S7Figure S9: Image of a film of the polyurethane SPU1 .........................................................................S7Figure S10:VT-FTIR spectra of SPU1, F1 and F2. .............................................................................S8Figure S11: Plots of the absorbance peak areas of VT-FTIR spectra………………………………...S9Figure S12 : Rheology profileof the Krasol HLBH-P2000 2 . .............................................................S9Figure S13: SAXS patterns of cast films of the SPU1, PEG, F1 and F2...........................................S10Table S1: Dimensions of printed samples……………………………………………………….. …S10 Table S2: Masses of printed samples……………………………………………………………..…S11 Figure S14: SAXS patterns of cast and 3D printed formulations F1 and F2…………………….…S11 Figure S15: Images of the printed F1) showing creep over 7 days…………………………….……S12Figure S16: Variation of moduli with frequency for F1 and F2 over 7 days .....................................S12Figure S17: Variation of moduli vs. frequency for drop cast and 3D printed F1 and F2...................S13Figure S18: Stress-strain curves of the Printed F1 and F2 upon aging at ambient condition. ..........S14Figure S19: Comparison of the tensile properties of printed F1 and F2 over 7 days.........................S14Table S3: Summary of tensile testing for creep in printed bars …………………………….……….S15Table S4: Predicted paracetamol release behaviour of the printed implants…………….………..…S15Figure S20: Images of the printed prototype implants F1 and F2 after dissolution testing ...............S15Figure S21: HPLC calibration curve for release study.......................................................................S16References..………………………………………………………………………………………….S16

Electronic Supplementary Material (ESI) for Polymer Chemistry.This journal is © The Royal Society of Chemistry 2020

S2

Synthesis of N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine 1

H2N

O

+

O

NO2

N

NO2O

NH

NO2O

EtOH

50 °C

NaBH4

1

N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine 1 was synthesised in accordance to the

previously reported procedure.S1 Briefly; to a stirred solution of 4-nitrobenzaldehyde (4.00 g, 26.45

mmol) in ethanol (50 mL) was added 4-methoxybenzylamine (4.38 g, 31.92 mmol) and resulting

mixture stirred at 50 °C for 18 hours. Ethanol was removed in vacuo before the crude solid was

passed through silica plug using ethyl acetate as the eluant. The resultant imine intermediate was then

dissolved in methanol (50 mL) and stirred with sodium borohydride (39. 68 mmol, 1.5 eq., 1.50 g) for

4 hours at room temperature. The solvent was subsequently removed in vacuo after which water was

added and the product isolated by extraction with ethyl acetate (3 × 50 mL) to yield an orange oil

(7.18 g, 84%). IR (ATR) ν/cm-1: 3098, 3024, 3008, 2963, 2932, 2913, 2835, 1676, 1639, 1605, 1600,

1580, 1510, 1443, 1440, 1417, 1378, 1340, 1298, 1295, 1245, 1219, 1179, 1144, 1106; 1H NMR (400

MHz, CDCl3) 𝛿 ppm: 8.18 (2H, appt.d, AA’XX’, CCHCHCNO2), 7.52 (2H, appt.d, AA’XX’,

CCHCHCNO2), 7.24 (2H, appt.d, AA’BB’, CCHCHCOCH3), 6.89 (2H, appt.d, AA’BB’,

CCHCHCOCH3), 3.90 (2H, s, NHCH2ArNO2), 3.81 (3H, s, ArOCH3), 3.75 (2H, s,

NHCH2ArOCH3), 1.60 (s, br, NH), 13C NMR (100 MHz; CDCl3) 𝛿 ppm: 159.1, 158.9, 149.0, 141.7,

130.5, 129.4, 128.9, 123.8, 114.1, 64.6, 55.3.

S3

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5δ(ppm)

1.14

2.03

3.05

2.01

2.01

2.10

2.00

2.00

-0.00

1.72

3.753.803.89

6.876.89

7.247.26

7.517.53

8.168.19

Figure S1: 1H NMR spectrum of N-(4-methoxybenzyl)-1-(4-nitrophenyl)methanamine.

0102030405060708090100110120130140150160170180δ(ppm)

52.21

52.67

55.31

76.75

113.89

123.61

128.67

129.32

131.86

147.01

148.25

158.81

Figure S2: 13C NMR of N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine.

S4

Synthesis of the N-(4-Methoxybenzyl)-1-(4- nitrophenyl)methanamine capped polyurethane

(SPU1).S1

The polyol was dried in vacuum oven at 120 °C under partial vacuum for 1 hour prior to use. Krasol

HLBH-P 2000 2 (20 g, 9.5 mmol) was mixed with 4,4′-Methylenebis(phenyl isocyanate) 3 (5.02 g,

20.1 mmol) in the bulk for 3 hours at 80 °C under N2. The resulting mixture was then dissolved in

anhydrous THF (150 mL) and cooled to room temperature. To the pre-polymer 4 was added N-(4-

methoxybenzyl)-1-(4-nitrophenyl)methanamine 1 (5.57 g, 20.46 mmol) and the solution stirred for 2

hours at 50 °C. The solution was concentrated in vacuo and the product isolated with repeated

precipitations from THF in methanol (3 × 1 L) to yield the product SPU1 as a light orange coloured

solid (22.6 g, 75%). IR (ATR) ν/cm-1: 3331, 2957, 2921, 2851, 1644, 1596, 1512, 1462, 14)12, 1342,

1304, 1241, 1174, 1108, 1031, 972, 813, 777, 735, 700 1H NMR (400 MHz, CDCl3) 𝛿 8.20 (4H,

appt.d, AA’XX’, CCHCHCNO2), 7.50 (4H, appt.d, AA’XX’, CCHCHCNO2), 7.17 (4H, appt.d,

AA’BB’, CCHCHCOCH3), 7.14–6.98 (16H, m, Ar), 6.89 (4H, appt.d, AA’BB’, CCHCHCOCH3),

6.50 (2H, br, NHC(O)O), 6.32 (2H, NHC(O)NH), 4.75 (5H, s, NCH2ArNO2), 4.47 (5H, s,

NCH2ArOCH3), 4.20 - 4.10 (4H, m, CH2CH2OC(O)NH), 3.86 (4H, m, ArCH2Ar), 3.81 (6H, s,

ArOCH3), 2.03–0.65 (407H, m, ((CH2)4)n. 13C NMR (400 MHz, CDCl3) 𝛿 159.6, 155.8, 147.4, 145.5,

136.4, 135.9, 129.4, 128.4, 128.1, 127.8, 124.0, 120.2, 114.7, 67.9, 55.4, 50.7, 40.5, 39.3, 37.8, 36.1,

33.8, 32.7, 30.7, 30.2, 29.8, 26.8, 25.7, 10.9; GPC (THF): Mn = 6100 Da, Mw = 12600 Da, Đ = 2.06;

DSC: Tg = -47.6 °C, 19.45 °C.

S5

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0δ(ppm)

228.67

13.90

4.00

4.82

5.02

2.04

1.69

25.47

4.73

5.34

0.000.810.820.840.860.880.890.960.980.991.021.041.061.071.211.231.251.321.331.351.371.371.431.451.561.591.661.671.821.891.982.003.723.723.773.783.813.823.843.863.873.883.90

4.124.144.154.174.184.47

4.75

6.326.326.506.536.896.906.916.926.937.027.047.047.067.067.087.097.107.117.137.157.177.197.257.267.277.297.487.508.198.208.218.22

Figure S3: 1H NMR spectrum of the polyurethane SPU1.

0102030405060708090100110120130140150160δ(ppm)

10.88

25.87

26.78

29.76

30.21

30.66

33.24

36.11

38.36

38.87

40.53

50.65

55.36

67.98

114.70

120.22

124.01

127.81

128.09

128.44

129.41

136.58

145.52

147.43

155.84

159.56

Figure S4: 13C NMR spectrum of the polyurethane SPU1.

S6

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5δ(ppm)

OO

22.5 65 12.5NH

O HN

ONH

HN

2

NO2

O

N

O

NO2

O

N

O

HOOH

22.5 65 12.5

Krasol HLBH-P2000 2

SPU1

NH

OO2N

Figure S5: Stacked 1H NMR spectra of the end-group (N-(4-Methoxybenzyl)-1-(4-nitrophenyl)methanamine 1, polymer 2, and SPU1, confirming the formation of the urethane and urea linkages.

85

90

95

100

600110016002100260031003600

T (%

)

Wavenumber (cm-1)

Figure S6: Infrared spectrum of SPU1.

S7

4 6 8 10 12 14 16 18 20RT (min)

Mw = 12600

Mn = 6100

Đ = 2.06

4 6 8 10 12 14 16 18 20RT (min)

Mw = 4300

Mn = 3900

Đ = 1.1

A B

Figure S7: GPC eluograms of A) SPU1 and B) Krasol HLBH-P2000. The regions used for molecular weights calculations are shown between the hashed lines. Analysis was carried out in THF against PS standards. The small peaks above 18 min are system peaks.S2

-2

-1

0

1

-100 -50 0 50 100 150 200

Heat

Flo

w (W

/g)

Temperature (°C)

Exo up

Tg = -47.85 °C

Tg = 19.45 °C

A

S8

-3.5

-2.5

-1.5

-0.5

0.5

-100 -50 0 50 100 150 200

Heat

Flo

w (W

/g)

Temperature (°C)

Tg1 = -46.6 °C

Tg2 = -0.6 °C

Exo up

Tm = 57.6 °C-3.5

-2.5

-1.5

-0.5

0.5

-100 -50 0 50 100 150 200

Heat

Flo

w (W

/g)

Temperature (°C)

Tg1 = -46.3 °C

Tg2 = -6.3 °CTm = 57.1 °C

Exo up

-4

-3

-2

-1

0

1

-100 -50 0 50 100 150 200

Heat

Flo

w (W

/g)

Temperature (°C)

Tg1 = -48.2 °CTg2 = 1.76 °C

Exo up

-3.5

-2.5

-1.5

-0.5

0.5

-100 -50 0 50 100 150 200

Heat

Flo

w (W

/g)

Temperature (°C)

Tg1 = -46.0 °C

Tg2 = -9.9 °CTm = 57.1 °C

Exo up

B.1

B.2

C.1

C.2

Figure S8: Differential Scanning Calorimetry of A) SPU1, B) F1 1: cast and 2: printed, C) F2 1: cast and 2: printed showing all thermal transitions. Shown in the graphs are the second cycle of the 3 cycles experiment.

Figure S9: Image of a film of the polyurethane SPU1, cast from THF.

S9

0

0.05

0.1

0.15

0.2

0.25

31003200330034003500

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

0

0.02

0.04

0.06

0.08

0.1

31003200330034003500

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

0

0.02

0.04

0.06

0.08

31003200330034003500

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

0

0.05

0.1

0.15

0.2

0.25

0.3

1630168017301780

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

0

0.05

0.1

0.15

0.2

163016801730

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

0

0.1

0.2

0.3

0.4

0.5

0.6

1630168017301780

Abso

rban

ce (

arb.

u.)

Wavenumber (cm-1)

20 °C 30 °C 40 °C 50 °C60 °C 70 °C 80 °C 90 °C100 °C 110 °C 120 °C 130 °C140 °C 150 °C 160 °C

B.3

B.2

B.1A.1

A.2

A.3

Figure S10: VT-FTIR spectra on heating (20 °C – 160 °C) of 1) SPU1, 2) F1 and 3) F2. Shown are A) NH stretching region and B) carbonyl stretching region. Arrows show the shift from hydrogen bonding towards free species.S3

S10

5

8

11

14

17

20

23

10 60 110 160

Area

(arb

. u.)

Temperature (°C)

H bonded NH

Free carbonyl

H bonded carbonyl

1

3

5

7

9

11

13

10 60 110 160

Area

(arb

. u.)

Temperature (°C)

H bonded NHFree carbonylH bonded carbonyl

1

2

3

4

5

6

7

8

10 60 110 160

Area

(arb

. u.)

Temperature (°C)

H bonded NHFree cabonylH bonded carbonyl

A B

C

Figure S11: Plots of the absorbance peak areas corresponding to hydrogen bonding NHs, in addition to free and hydrogen bonded carbonyls for A) SPU1, B) F1 and C) F2 from IR spectroscopy. Areas were calculated by fitting Gaussian distributions to IR absorbances in order to deconvolute the signals.

10-5

10-3

10-1

101

103

105

0

30

60

90

0 50 100 150

Phas

e An

gle

(°)

Mod

ulus

(Pa)

Temperature °C

G'

G"

Phaseangle

Figure S12 : Rheological behaviour of the viscous liquid, hydrogenated polybutadiene (Krasol HLBH-P2000) 2 used as the starting material in the synthesis of SPU1.

S11

Figure S13: SAXS patterns of cast films of the SPU1, PEG (20 kDa), F1 and F2.

Table S1: The dimensions and calculated percentage deviations of each print. Deviation = 100 × (individual print value-mean value) / (mean value)

Formulation F1

Sample Number Length (mm) Width (mm) Thickness (mm) Length deviation width deviation Thickness

deviation 1 29.63 3.20 2.55 0.16% 2.79% 0.43% 2 29.87 3.20 2.52 0.97% 2.79% -0.75% 3 29.64 3.10 2.57 0.20% -0.42% 1.22% 4 29.68 3.05 2.53 0.33% -2.02% -0.35% 5 29.68 3.15 2.55 0.33% 1.19% 0.43% 6 29.20 2.99 2.55 -1.29% -3.95% 0.43% 7 29.50 3.10 2.51 -0.28% -0.42% -1.14% 8 29.40 3.09 2.54 -0.62% -0.74% 0.04% 9 29.80 3.10 2.52 0.74% -0.42% -0.75%

10 29.42 3.15 2.55 -0.55% 1.19% 0.43% STD 0.19 0.06 0.02

Mean 29.58 3.11 2.54

S12

Table S2: The masses and calculated percent deviation from the mean for each print. Percent deviation = 100 × (implant mass − mean implant mass) / (mean implant mass)

Formulation F1

Sample Number Mass (g) Mass Deviation

1 0.2252 0.51% 2 0.2272 1.38% 3 0.2261 0.93% 4 0.2294 2.39% 5 0.2283 1.89% 6 0.2187 -2.41% 7 0.2190 -2.27% 8 0.2222 -0.85% 9 0.2223 -0.81% 10 0.2224 -0.76%

Mean 0.224

Formulation F2

Sample Number Mass (g) Mass Deviation

1 0.2316 9.47% 2 0.218 3.04% 3 0.2002 -5.38% 4 0.2125 0.44% 5 0.2104 -0.55% 6 0.2222 5.02% 7 0.1990 -5.94% 8 0.1941 -8.28% 9 0.2136 0.98% 10 0.2141 1.21%

Mean 0.212

Figure S14: A comparison of SAXS patterns from cast and 3D printed formulations F1 and F2.

S13

Printed bar day 0 Printed bar day 7

Figure S15: Images of the printed formulation F1 showing creep over 7 days.

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

A

B

C

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

10-2 10-1 100 101 102104

105

106

107

Mod

ulus

(Pa)

Frequency (Hz)

G'

G"

Figure S16: Variation of moduli with frequency for F1 (left) and F2 (right) over A) 0, B) 3 and C) 7 days aging period at ambient condition.

S14

10-2 10-1 100 101 102104

105

106

107El

astic

Mod

ulus

(Pa)

Frequency (Hz)

Cast film

Printed day 0

Printed day 3

Printed day 7

10-2 10-1 100 101 102104

105

106

107

Elas

tic M

odul

us (P

a)

Frequency (Hz)

Cast film

Printed day 0

Printed day 3

Printed day 7

A B

10-2 10-1 100 101 102104

105

106

Visc

ous

Mod

ulus

(Pa)

Frequency (Hz)

Cast film

Printed day 0

Printed day 3

Printed day 7

10-2 10-1 100 101 102104

105

106

Visc

ous

Mod

ulus

(Pa)

Frequency (Hz)

Cast filmPrinted day 0Printed day 3Printed day 7

Figure S17: Variation of elastic and viscous modulus vs. frequency at 25 °C for drop cast and 3D printed disks in A) F1 and B) F2.

S15

0.00

0.05

0.10

0.15

0.20

0 200 400 600

Stre

ss (N

.mm

-2 )

Strain (%)

0

0.04

0.08

0.12

0 100 200 300 400 500 600

Stre

ss (N

.mm

-2)

Strain (%)

0.00

0.04

0.08

0.12

0.16

0 200 400 600 800

Stre

ss (N

.mm

-2)

Strain (%)

0.00

0.05

0.10

0.15

0 200 400 600 800 1000

Stre

ss (N

.mm

-2)

Strain (%)

0.00

0.04

0.08

0.12

0.16

0 500 1000

Stre

ss (N

.mm

-2)

Strain (%)

0.00

0.03

0.06

0.09

0.12

0 200 400 600 800 1000

Stre

ss (N

.mm

-2)

Strain (%)

A

B

C

Figure S18: Stress-strain curves of the Printed F1 (left) and F2 (right) upon aging at ambient condition for A) 0, B) 3 and C) 7 days. Each graph shows three replications.

0

0.5

1

1.5

2

2.5

3

3.5

F1 F2

Mod

. (M

Pa)

As cast

Printed day 0

Printed day 3

Printed day 7

0

0.05

0.1

0.15

0.2

0.25

F1 F2

Mod

. (M

Pa)

As cast

Printed day 0

Printed day 3

Printed day 7

A B

Figure S19: Bar charts comparing the A) ultimate tensile strength and B) Young’s modulus of printed formulations F1 and F2 over 7 days.

S16

Table S3: Summary of tensile testing comparing cast formulations (F1 and F2) and creep printed bars.

F1 F2

Cast Day 0 Day 3 Day 7 Cast Day 0 Day 3 Day 7

Young’s

Modulus (MPa)

1.87 ±

0.10

1.95 ±

0.57

2.52 ±

0.15

2.80 ±

0.11

1.52 ±

0.13

1.91 ±

0.50

2.09 ±

0.34

1.71 ±

0.22

Ultimate Tensile

Strength (MPa)

0.20 ±

0.01

0.16 ±

0.02

0.15 ±

0.01

0.14 ±

0.01

0.20 ±

0.03

0.11 ±

0.02

0.10 ±

0.01

0.11 ±

0.01

Table S4: The predicted paracetamol release behaviour from the implants based on fitting the dissolution data to Zero order, First order and Korsmeyer-Peppas release models.

F1 F2

Model

Predicted time to 100%

release (month)

R2 Slope y-intercept

Predicted time to 100%

release (month)

R2 Slope y-intercept

Zero Order 3.99E+01 0.9285 3.46E-03 1.84E+00 6.54E+01 0.9975 2.11E-03 1.58E+00

First Order 8.48E+00 0.9062 7.02E-04 2.68E-01 4.91E+00 0.999 1.18E-03 4.64E-01

Korsmeyer-Peppas 1.33E+12 0.9901 1.31E-01 8.82E-02 2.88E+18 0.9604 9.00E-02 1.87E-01

A B

Figure S20: Images of the printed paracetamol bars, F1 (A) and F2 (B), after dissolution testing (7 days) at 37 °C.

S17

y = 3.8444E+01x - 2.7564E-01R² = 9.9998E-01

0

20

40

60

80

100

0 0.5 1 1.5 2 2.5

Peak

Are

a (m

AU*s

)

Concentration (ppm)

Figure S21: HPLC calibration curve for paracetamol in 0.05 M phosphate buffer (pH 6.8).

References

S1 A. Feula, A. Pethybridge, I. Giannakopoulos, X. Tang, A. Chippindale, C. R. Siviour, C. P. Buckley, I. W. Hamley and W. Hayes, Macromolecules, 2015, 48, 6132–6141.

S2 D. Held and F. Gores, The Column, 2019, 2, 17–21. http://www.chromatographyonline.com/tips-tricks-gpcsec-system-peaks-or-ghost-peaks-gpcsec, (accessed 29 March 2020)

S3 D. Hermida-Merino, A. T. Slark, H. M. Colquhoun, W. Hayes and I. W. Hamley, Polym. Chem., 2010, 1, 1263–1271.