poly(methacrylic acid) complexation of amphotericin b

Les et al Electronic supporting information

1

Poly(methacrylic acid) complexation of amphotericin B

Karolina Alicja Les1,2, Abeer Mohamed Ahmed2,3, Sibu Balan1,2, Ji-won Choi1, Denis

Martin4, Vanessa Yardley5, Keith Powell1, Antony Godwin1 and Steve Brocchini2,3 1PolyTherics Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK. 2UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK. 3NIHR Biomedical Research Centre, Moorfields Eye Hospital and UCL Institute of Ophthalmology, London, EC1V 9EL, UK. 4Drugs for Neglected Diseases Initiative, 15 Chemin Louis-Dunant, 1202 Geneva, Switzerland. 5Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, WC1E 7HT, UK

Supplementary experimental information

Preparation of AmB-PMAA complexes from PMOSu

Poly(N-methacryloxysuccinimide)1 (PMOSu) 4 (60 mg, 0.3 mmol of the repeat unit)

was dissolved in stirred DMSO (1.2 mL) overnight in a 50 mL round bottom flask. To

this stirred solution was added a solution of AmB (48 mg, 0.05 mmol) in DMSO

(1.4 mL). After 2 min, an aqueous solution of sodium hydroxide (0.656 mL 1 M

NaOH and 110 µL H2O) was added dropwise to the PMOSu/AmB solution followed

by the immediate addition of pure water (6.7 mL). The resulting solution was left to

stir at room temperature for 1 h. Uncomplexed AmB was then removed by 24 h

dialysis against 1 L of pure water using a Visking dialysis membrane (MW12-14 kDa,

Medicell International). During dialysis, the dialysate was changed six times. The

dialysed solution of the complex was filtered using a 0.2 µm Minisart® syringe filter

(Fisher, Cat. No. FDP-620-030U) and then freeze-dried to afford a red coloured

powder (mass 96.7 mg). Samples were protected from light by wrapping in

aluminium foil at all times during preparation and storage.

Reference

(1) A. Godwin, M. Hartenstein, A. Müller and S. Brocchini, Angew. Chem. Int. Ed. Engl. 2001, 40(3), 594-597.

Electronic Supplementary Material (ESI) for Polymer ChemistryThis journal is © The Royal Society of Chemistry 2013


2

Supplementary Figures

Figure S1

A

B

C

Figure S1. The HPLC chromatogram for Funigzone (AmB-D) (A) was obtained using the amount of AmB-D necessary to have a 60 µg/mL concentration of AmB and the chromatograms for (B) and (C) were obtained using 60 µg/mL of material. AmB eluted at ~24.5 minutes.

Fungizone (AmB-D)

PRIME [version 4.2.0] - FILE: C:\PW4\Sibu-AmBpurity-2\0005_Sibu-AmBpurity-2.pw4Name: (unnamed)Description:

Injection Time: 25 June 2007 at 23:08:48 [Active Status]Type: AUTOINJ Injection Number: 5 Channel: Channel A Acquisition Rate: 4HzMethod File: C:\PW4\Sibu-AmBpurity-2\0001_Sibu-AmBpurity-2.mth Baseline noise: (not calculated)Standard File: (none) Sequence File: C:\PW4\Sibu-AmBpurity-2\Sibu-AmBpurity-2.seq

mV

Minutes

-9.1

18.7

46.5

74.4

102.2

130.0

7.5 10.5 13.6 16.7 19.7 22.8 25.8 28.9 31.9 35.0

1

9.36

3

2

10.8

25

3

16.5

04

424.708

Peak RT Area %Ar Conc.(Ar) Height M Units Name 1 9.363 1595.500 11.43 Not Calculated 49.434 0 2 10.825 220.210 1.58 Not Calculated 4.154 0 3 16.504 471.710 3.38 Not Calculated 8.515 1 4 24.708 11671.040 83.61 Not Calculated 106.188 1

Page 1 of 1 - Jun 26, 2007 10:34 AM



mV

Minutes

-9.1

18.7

46.5

74.4

102.2

130.0

7.5 10.5 13.6 16.7 19.7 22.8 25.8 28.9 31.9 35.0

1

9.36

3

2

10.8

25

3

16.5

04

424.708

Peak RT Area %Ar Conc.(Ar) Height M Units Name 1 9.363 1595.500 11.43 Not Calculated 49.434 0 2 10.825 220.210 1.58 Not Calculated 4.154 0 3 16.504 471.710 3.38 Not Calculated 8.515 1 4 24.708 11671.040 83.61 Not Calculated 106.188 1

Page 1 of 1 - Jun 26, 2007 10:34 AM



mV

Minutes

-9.0

18.8

46.6

74.4

102.2

130.0

8.6 11.5 14.5 17.4 20.3 23.3 26.2 29.1 32.1 35.0

1

9.35

0

2

16.4

54

324.575

Peak RT Area %Ar Conc.(Ar) Height M Units Name 1 9.350 156.489 1.44 Not Calculated 4.492 1 2 16.454 610.320 5.62 Not Calculated 12.380 1 3 24.575 10097.092 92.94 Not Calculated 108.370 1

Page 1 of 1 - Jun 26, 2007 10:36 AM



mV

Minutes

-9.0

18.8

46.6

74.4

102.2

130.0

8.6 11.5 14.5 17.4 20.3 23.3 26.2 29.1 32.1 35.0

1

9.35

0

2

16.4

54

324.575


Page 1 of 1 - Jun 26, 2007 10:36 AM

Sigma Aldrich AmB-Batch A4888



mV

Minutes

-9.0

18.8

46.6

74.4

102.2

130.0

10.3 13.1 15.8 18.5 21.3 24.0 26.8 29.5 32.3 35.0

1

9.32

9

2

19.1

37

324.425


Page 1 of 1 - Jun 26, 2007 10:37 AM

Batch HAP0611501



mV

Minutes

-9.0

18.8

46.6

74.4

102.2

130.0

10.3 13.1 15.8 18.5 21.3 24.0 26.8 29.5 32.3 35.0

1

9.32

9

2

19.1

37

324.425


Page 1 of 1 - Jun 26, 2007 10:37 AM



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Figure S2

(A)

(B)

Figure S2. (A) Overlay of the FT-IR spectrum of PMAA and PMAA-Na. (B) Comparison of FT-IR spectra of PMAA protonated using salt precipitation method (1) and dialysis method (2).

PMAA-Na

PMAA

1

2



4

Figure S3

Figure S3. Photographic images of freeze-dried AmB-PMAA complex.

Figure S4

A B

Figure S4. AmB Calibration curves. (A) AmB in 50% methanol measured by UV-Vis and (B) RP-HPLC where elution was conducted with a 1 mL/min gradient starting from 30% acetinitrile in 25 mM EDTA to 100% acetonitrile (20 min) using a SUPERCOSIL™ LC-18-DB column. Both detected at 409 nm and N= 3.

Figure S5

Figure S5. Image of the AmB-PMAA complex 3 by transmission electron microscopy (Direct Mag 93000). The AmB-PMAA complex contained 35 wt% AmB and was frabricated from PMAA with a molecular weight of 31.1 kDa.



5

Supplementary Tables

Table S1

Batch PVP:PMAA (mol:mol)

AmB in complex [Wt%]

Amount PMAA used

(mg)

Amount PVP used

(mg)

Amount AmB used

(mg)

1 0 2 0 12.5 12.5 2 0.5 19 20 26 20 3 1 35 20 0 20

Table S1. The AmB content in a complex prepared when using PVP (58 kDa) as the only polymer for complexation (Batch 1) was very low. Increased AmB loading was observed when the relative amount of PMAA (31.1 kDa) was also increased.

Table S2

AmB (mg)

PMAA (mg)

AmB content-

UV (wt%)

AmB loading-HPLC (wt%)

AmB complexation

efficiency (%)

Yield [%]

52 70 25 23 50 81 112 70 36 34 40 64 150 70 41 46 44 70 131 100 36 -- 52 77 252 200 33 37 46 74

Table S2. AmB content, complexation efficiency and complex preparation yields for selected samples prepared using different ratios of AmB to PMAA at different scales of complexation.

Table S3

PMAA MWt (kDa)

AmB content (wt%)

7.5 35 ± 2 31.1 33 ± 5 100 28 ± 3 7.5* 30 31.1* 32 ± 3 65.5* 31 ± 1 100* 32

Table S3. The effect of the MW of PMAA used to prepare a complex and the AmB content in the final complex. All samples, prepared in triplicates, were prepared with a targeted AmB content of 30 wt% using the same weight ratios of AmB to PMAA. *PVP present.


poly(methacrylic acid) complexation of amphotericin b

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