SUPPLEMENTARY INFORMATION

doi: 10.1038/nchem.1012
Supplementary Information

Biodegradable Nanostructures with Selective Lysis of Microbial Membranes

Fredrik Nederberg, Ying Zhang, Jeremy P.K. Tan, Kaijin Xu, Huaying Wang, Chuan
Yang, Shujun Gao, Xin Dong Guo, Kazuki Fukushima, Lanjuan Li, James L. Hedrick and
Yi-Yan Yang

Critical micelle concentration (CMC) determination. CMC is an important parameter,

above which an amphiphilic macromolecule forms core/shell structured nanoparticles (i.e.

micelles). The CMC values of polymers in DI water and the buffer were estimated by

fluorescence spectroscopy using pyrene as a probe. The fluorescence spectra were

recorded by a LS 50B luminescence spectrometer (Perkin Elmer, U.S.A.) at room

temperature. Aliquots of pyrene in acetone solution (6.16×10-5 M, 10 μL) were added to

containers and acetone was left to evaporate. Polymer solutions (1 mL) at varying

concentrations were added into the containers and left to equilibrate for 24 hours. The

final pyrene concentration in each sample was 6.16×10-7 M. The excitation spectra were

scanned from 300 to 360 nm at an emission wavelength of 395 nm. Both the excitation

and emission bandwidths were set at 2.5 nm. The intensity (peak height) ratios of I337/I334

from the excitation spectra were analyzed as a function of polymer concentration. The

CMC was taken from the intersection between the tangent to the curve at the inflection

and tangent of the points at low concentrations.

Minimal inhibitory concentration (MIC) determination. Bacillus subtilius and

Staphylococcus aureus were obtained from ATCC, and grown in tryptic soy broth at

37°C. Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis and

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Cryptococcus neoformans were extracted from patients’ phlegm, and kindly provided by

Y. S. Yu, Department of Infectious Diseases, The First Affiliated Hospital, College of

Medicine, Zhejiang University, P. R. China. The clinical samples were grown in Mueller-

Hinton broth at 37°C. The MICs of the polymers were measured using a broth

microdilution method. Briefly, 50 μL of polymer solution with various concentrations

was placed into each well of 96-well plates. 50 μL of microorganism solution at a

concentration, which gave an optical density reading of ~ 0.1-0.2 at 600 nm, was added

to each well. The optical density readings of microorganism solutions were measured as a

function of time. The MIC was taken at the concentration, at which no growth was

observed with the unaided eyes and microplate reader (Bio-Teck Instruments, Inc), and in

the growing phase of the microorganisms. Broth containing cells alone was used as

control. Antimicrobial agents used in clinic for treatment of infections caused by these

types of microbes, i.e. vancomycin for Bacillus subtilis, Staphylococcus aureus,

methicillin-resistant Staphylococcus aureus and Enterococcus faecalis and amphotericin

B for Cryptococcus neoformans, were employed as a comparison. The tests were

repeated at least three times.

Colony assay. The counts of live microbes in the treated microbial samples were

estimated by a colony formation assay. After 8 or 24 (in the case of Cryptococcus

neoformans) hours of incubation with polymer and conventional antimicrobial samples of

various concentrations, microbial solution was diluted with an appropriate dilution factor

and paced on an agar plate (LB Agar from 1st Base). The plate was then incubated

overnight at 37°C. The number of colony-forming units (CFU) was counted and

expressed as CFU/mL. Percentage of the counts of live microbe in the treated sample as

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compared to the counts in the control sample without any treatment was estimated as a

function of polymer/antimicrobial concentration.

Hemolysis assays. Fresh mouse red blood cells were washed with PBS for three times.

100 μL of red blood cell suspension in PBS (4% in volume) was placed in each well of

96-well plates and 100 μL of polymer solution was added to each well. The plates were

incubated for one hour at 37°C. The cell suspensions were taken out and centrifuged at

1000g for 5 minutes. Aliquots (100 μL) of supernatant were transferred to 96-well plates,

and hemoglobin release was monitored at 576 nm using a microplate reader (Bio-Teck

Instruments, Inc). The red blood cell suspension in PBS was used as negative control.

Absorbance of wells with red blood cells lysed with 0.5% Triton X-100 was taken as

100% hemolysis. Percentage of hemolysis was calculated using the following formula:

Hemolysis (%) = [(O.D.576nm in the nanoparticle solution-O.D.576nm in PBS)/(O.D.576nm in

0.5% Triton X-100 – O.D.576nm in PBS)]×100.

Transmittance electron microscopy (TEM). The morphologies of the microorganisms

before and after treatment with the micelles were observed under a JEM-1230

transmittance electron microscope (JEOL, Japan) using an acceleration voltage of 80 keV.

The microorganism solution (1.5 mL) was incubated with 0.5 mL of polymer 3 micelle

solution at lethal doses (10.8 µM for methicillin-resistant Staphylococcus aureus and 16.3

µM for Enterococcus faecalis and Cryptococcus neoformans) for 8 hours. The solution

was centrifuged at 5000 rpm for 10 minutes, and the supernatant was removed.

Phosphate buffer (pH7.0, 0.5 mL) containing 2.5% glutaraldehyde was added to the

microorganisms, and incubated overnight at 4°C for fixation. The sample was

washed three times with the phosphate buffer (15 minutes each), and then post-

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fixed with 1% OsO4 in the phosphate buffer (pH7.0) for one hour. The fixed

sample was washed three times in the phosphate buffer (15 minutes each), followed

by dehydration in a graded ethanol series.

The sample was incubated with the mixture of acetone and Spurr resin (1:1 in volume)

for one hour at room temperature, which was then transferred to 1:3 mixture of acetone

and Spurr resin for 3 hours, and to Spurr resin for overnight. Ultrathin sections (70-90

nm) were cut using a Reichert-Jung Ultracut E ultramicrotome, and post-stained with

uranyl acetate and lead citrate for 15 minutes each prior to TEM observations.

TEM image of the micelles were obtained using a FEI Tecnai G2 F20 electron

microscope with an acceleration voltage of 200 keV. To prepare the TEM sample, several

drops of the micelle solution were placed on a formvar/carbon coated 200 mesh copper

grid and left to dry under room temperature.

In vitro polymer degradation. In vitro degradation of polymer 1 and polymer 3 was

conducted in DI water at 37°C. Polymer samples were taken periodically and freeze-dried

prior to GPC analysis.

In vivo toxicity studies. Animal studies were performed according to protocols approved

by IBM’s Animal Care and Use Committee, and the Singapore Biological Research

Center (BRC)’s Institutional Animal Care and Use Committee. Female Balb/c mice (6-7

weeks, 18-22 g) were used in all experiments for studies of LD50 and toxicity to the

major organs liver and kidney, and electrolyte balance in the blood.

LD50 was determined according to the Up-and-Down-Procedure described in OECD

Guidelines for the Testing of Chemicals (OECD 425). Twenty mice were observed for 7

days and randomly marked to permit individual identification. Polymer 1 was dissolved

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in PBS (pH 7.4), and given to mice via tail vein injection at designed doses (i.e. 5.5, 17.5,

56.0 and 179.2 mg/kg, 0.15 mL). Mortality was monitored for 14 days post-treatment,

and LD50 was determined using the maximum likelihood method.

For the evaluation of the acute toxicity, two groups of 10 mice each received intravenous

injection of polymer 1 at 12 mg/kg in 150 µL of sterilized saline. 10 mice were sacrificed

at 48 hours and the rest mice at 14 days to collect blood samples for analysis of ALT,

AST, creatinine, urea nitrogen, sodium ion and potassium ion levels.

Table S1 Physicochemical properties of polymers

Polymer Mn m/n* PDI† CMC Size Zeta
(g/mol) (µg/mL) in (nm) potential
DI water (mV)
1 6200 10/30 1.15 35.5 43±7 47±3
2 7800 10/45 1.26 15.8 402±21 65±5
3 9200 20/30 1.25 70.8 198±9 60±3
*
Estimated from 1H NMR analysis. † Obtained from gel permeation chromatography
(GPC) analysis using THF as solvent and calibrated against PS standards. Analysis done
on the intermediate copolymers (PTMC-PMTCpropylCl) prior to quaternation.

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A
t=0
t = 8 hrs.
t = 1 day
t = 14 days

10 15 20 25 30 35 40 45 50 55
Elution Time(min.)

t=0
B t = 8 hrs
t = 24 hrs
t = 15 days

10 15 20 25 30 35 40
Elution Time (min.)

Fig. S1 GPC analysis results of degrading polymer 1 (A) and polymer 3 (B) at different
time points.

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1.8
A
1.6
1.4
337 /I 334 )

1.2
Intensity ratio (I

1.0
0.8
0.6
0.4 CMC = 35.5 mg/L
0.2
0.0
-1.0 0.0 1.0 2.0 3.0 4.0
lg concentration (mg/L)

1.8
1.6 B

1.4
337 /I 334 )

1.2
Intensity ratio (I

1.0
0.8
0.6
0.4
CMC = 17.8 mg/L
0.2
0.0
-1.0 0.0 1.0 2.0 3.0 4.0
lg concentration (mg/L)

Fig. S2 Plot of I337/I334 ratio as a function of logarithm of polymer 1 concentration (lg C,

mg/L) in DI water (A) and buffer (B).

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1.8
1.6 A
1.4
Intensity ratio (I 337 /I334 ) 1.2
1.0
0.8
0.6
0.4
CMC = 15.8 mg/L
0.2
0.0
-1.0 0.0 1.0 2.0 3.0 4.0
lg concentration (mg/L)

1.8
1.6 B
Intensity ratio (I 337 /I334 )

1.4
1.2
1.0
0.8
0.6
0.4 CMC = 11.2 mg/L
0.2
0.0
-1 0 1 2 3 4
lg concentration (mg/L)

Fig. S3 Plot of I337/I334 ratio as a function of logarithm of polymer 2 concentration (lg C,

mg/L) in DI water (A) and buffer (B).

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1.6
1.4 A

Intensity ratio (I 337 /I334 ) 1.2

1.0
0.8
0.6
0.4 CMC = 70.8 mg/L
0.2
0.0
-1.0 0.0 1.0 2.0 3.0 4.0
lg concentration (mg/L)

1.8
1.6 B
Intensity ratio (I 337 /I334 )

1.4
1.2
1.0
0.8
0.6
0.4
CMC = 28.2 mg/L
0.2
0.0
-1.0 0.0 1.0 2.0 3.0 4.0
lg concentration (mg/L)

Fig. S4 Plot of I337/I334 ratio as a function of logarithm of polymer 3 concentration (lg C,

mg/L) in DI water (A) and buffer (B).

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1.0
0.9 Control
0.8 1.0
7.8
2.0
15.6
0.7
O.D. at 600 nm

4.1
31.3
0.6 8.0 μM
62.5
0.5 125
16.0
0.4 32.0
250
0.3 64.0
500
0.2
0.1
0.0
0 2 4 6 8 10
Time (Hours )

Fig. S5 Dose-dependent growth inhibition of Bacillus subtilis in the presence of

polymer 2 (MIC: > 64.0 µM). The data are expressed as mean and standard
deviations of at least three replicates. The standard deviation is shown by the error
bars. OD, optical density.

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A
0.8
Control
0.7 3.2
20
0.6 4.9
30
O.D. at 600 nm

6.5 μM
40
0.5 9.7
60
0.4 12.9
80
16.2
100
0.3
0.2
0.1
0
0 3 6 8
Time (Hours)
B
1.2
Control
1.0 20
3.2
30
4.9
O.D. at 600 nm

0.8 40
6.5 μM
9.7
60
0.6 80
12.9
100
16.2
0.4

0.2

0.0
0 2 4 6 8
C Time (Hours)
1.0
0.9 Control
2.6
0.8 5.1
O. D. at 600 nm

0.7 7.7 µM
0.6 10.3
0.5 12.9
16
0.4 32.1
0.3
0.2
0.1
0.0
0 2 4 6 8
Time (Hours)

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D
0.7
Control
0.6 2.6
5.1
0.5 7.7
O. D. at 600 nm

10.3 µM
0.4 12.9
16
0.3 32.1
64.1
0.2
0.1
0
0 2 4 6 8 22 24
Time (Hours)
E
0.6
Control
0.5 2.6
5.1
7.7
O. D. at 600 nm

0.4 10.3 µM
12.9
0.3 16
32.1
0.2 64.1

0.1

0
0 2 4 6 8 22 24
Time (Hours)

Fig. S6 Dose-dependent growth inhibition of Bacillus subtilis (A), Staphylococcus

aureus (B), methicillin-resistant Staphylococcus aureus (C), Enterococcus faecalis
(D) and Cryptococcus neoformans (E) in the presence of polymer 1 (MIC: 9.7, 6.5,
5.1, 16.0 and 16.0 µM respectively). The data are expressed as mean and standard
deviations of at least three replicates. The standard deviation is shown by the error
bars. OD, optical density.

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0.8
Control A
0.7 0.17
0.34
0.6 0.67
1.3 µM
O.D. at 600 nm

0.5
2.7
0.4 5.4
10.8
0.3

0.2

0.1

0.0
0 2 4 6 8
Time (Hours)
0.9 Control
0.084 B
0.8 0.17
0.7
0.34
0.67
0.6
1.3 µM
2.7
O.D. at 600 nm

0.5 5.4
10.8
0.4 21.5

0.3

0.2

0.1

0
0 2 4 6 8
Time (Hours)
0.8
Control C
0.084
0.7 0.17
0.34
0.6 0.67
1.3 µM
2.7
0.5 5.4
O.D. at 600 nm

10.8
0.4 21.5

0.3

0.2

0.1

0.0
0 2 4 6 8
Time (Hours)
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0.40
Control D
0.35 0.084
0.17
0.34
0.30 0.67
1.3 µM
O.D. at 600 nm

0.25 2.7
5.4
0.20 10.8
21.5
0.15

0.10

0.05

0.00
0 2 4 6 8
Time (Hours)
0.9
Control E
0.8 0.14
0.27
0.54
0.7 1.1
2.2 µM
0.6 4.3
8.6
O.D. at 600 nm

17.3
0.5 34.6
0.4

0.3

0.2

0.1

0.0
0 2 4 6 8 22 24
Time (Hours)
Fig. S7 Dose-dependent growth inhibition of Bacillus subtilis (A), Staphylococcus
aureus (B), methicillin-resistant Staphylococcus aureus (C), Enterococcus faecalis
(D) and Cryptococcus neoformans (E) in the presence of antimicrobials that are
commonly used in clinic (vancomycin for A-D; amphotericin B for E) (MIC: 1.3,
1.3, 1.3, 0.67 and 8.6 µM respectively). The data are expressed as mean and
standard deviations of at least three replicates. The standard deviation is shown by
the error bars. OD, optical density.

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120

Percentage of CFUtest/CFUcontrol
100

80

60

40

20

0
0 10 20 30 40 50
120 Polymer 1 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 10 20 30 40 50 60
120 Polymer 3 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 2 4 6 8 10 12
Vancomycin concentration (µM)

Fig. S8 Percentage of CFU of Staphylococcus aureus in the samples

treated with polymer 1, polymer 3 and vancomycin at various
concentrations for 8 hours as compared to the control sample without any
treatment.

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120

Percentage of CFUtest/CFUcontrol
100

80

60

40

20

0
0 10 20 30 40 50
120 Polymer 1 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 10 20 30 40 50 60

120 Polymer 3 concentration (µM)

Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 5 10 15 20 25
Vancomycin concentration (µM)
Fig. S9 Percentage of CFU of methicillin-resistant Staphylococcus aureus
in the samples treated with polymer 1, polymer 3 and vancomycin at
various concentrations for 8 hours as compared to the control sample
without any treatment.

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120

Percentage of CFUtest/CFUcontrol
100

80

60

40

20

0
0 10 20 30 40 50
120 Polymer 1 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 5 10 15 20 25 30
120 Polymer 3 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 2 4 6 8 10 12
Vancomycin concentration (µM)
Fig. S10 Percentage of CFU of Enterococcus faecalis in the samples
treated with polymer 1, polymer 3 and vancomycin at various
concentrations for 8 hours as compared to the control sample without any
treatment.

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120

Percentage of CFUtest/CFUcontrol
100

80

60

40

20

0
0 10 20 30 40 50

120
Polymer 1 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 5 10 15 20 25 30
120 Polymer 3 concentration (µM)
Percentage of CFUtest/CFUcontrol

100

80

60

40

20

0
0 2 4 6 8 10
Amphotericin B concentration (µM)
Fig. S11 Percentage of CFU of Cryptococcus neoformans in the samples
treated with polymer 1, polymer 3 and amphotericin B at various
concentrations for 24 hours as compared to the control sample without any
treatment.

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95

75
% of Hemolysis 55

35

15
3
1
-5
0 100 200 300 400 500 600
Polymer concentration (μg/mL)

Fig. S12 Hemolytic activity of polymer 1 and 3 as a function of concentration.

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