SUPPLEMENTARY INFORMATION Supplementary Information … · nm) were cut using a Reichert-Jung Ultracut E ultramicrotome, and post-stained with uranyl acetate and lead citrate for

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nnano.2009.153

nature nanotechnology | www.nature.com/naturenanotechnology 1

Supplementary Information

Self-assembled Cationic Peptide Nanoparticles as An Efficient Antimicrobial Agent

Lihong Liu, Kaijin Xu, Huaying Wang, Jeremy Tan P. K. , Weimin Fan, Subbu S.

Venkatraman, Lanjuan Li and Yi-Yan Yang

Critical micelle concentration (CMC) determination of CG3R6TAT

CMC is an important parameter, above which an amphiphilic macromolecule forms

core/shell structured nanoparticles (i.e. micelles). The CMC value of CG3R6TAT in DI

water was estimated by fluorescence spectroscopy using pyrene as a probe. Fluorescence

spectra were recorded by a Flourolog fluorescence spectrometer (Jobin Yvon Horiba) at

room temperature. Aliquots of pyrene solutions in acetone were added to 15 mL

volumetric flasks, and the acetone was allowed to evaporate. Peptide solutions (4mL)

with different concentrations ranging from 0.1 to 200.0 ppm were then added to the

flasks. The final pyrene concentration is 6.16×10-7 M. The solutions were equilibrated for

24 hours at 35°C. The excitation spectra were recorded from 300 to 360 nm with an

emission wavelength of 395 nm. Both excitation and emission bandwidths were set at 2.5

nm. The intensity ratios of I339 to I334 were plotted as a function of logarithm of peptide

concentration. The CMC value was taken from the intersection of the tangent to the curve

at the inflection with the horizontal tangent through the points at low concentrations.

1

© 2009 Macmillan Publishers Limited. All rights reserved.

2 nature nanotechnology | www.nature.com/naturenanotechnology

SUPPLEMENTARY INFORMATION doi: 10.1038/nnano.2009.153

0.55

0.60

0.65

0.70

0.75

0.80

0.85

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5Log C

I339

/I334

Figure S1 Plot of I339/I334 ratio as a function of logarithm of peptide concentration (Log C, mg/L) in DI water.

Hydrodynamic radius, radius of gyration and aggregation numbers

Dynamic light scattering (DLS) and static light scattering (SLS) experiments were

performed on a Brookhaven BI-200SM goniometer system (U.S.A.) to determine the

hydrodynamic radius (Rh), gyration of radius (Rg) and weight average molecular weight

(Mw) respectively. The light source is a power adjustable and vertically polarized 75 mW

HeNe ion laser with a wavelength of 633 nm. For SLS, a Zimm plot (Eq 1) was used to

analyze the experimental data and the refractive index increment (dn/dc) was measured

using a BI DNDC differential refractometer (Brookhaven, U.S.A.). Samples are

equilibrated for 10 minutes before measurements were taken. The aggregation number

(Nw) was determined by the quotient of the micelle Mw and the unimer Mw.

( ) cARn

MRKc g

w22

2222

23

2sin16

11+

⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜

⎝

⎛⎟⎠⎞

⎜⎝⎛

+=λ

θπ

θ

2

where K (= ( ) 42224 λπ ANdcdnn ) is an optical constant, R(θ) (= θsin2ts IrI ) is the

Rayleigh ratio, c is the concentration of the polymer solution in gram per milliliter, n is

the refractive index of the solvent, θ is the angle of measurement, λ is the wavelength of

the laser in vacuum and NA is the Avogadro’s constant. A plot of (Kc/R(θ)) against

(sin2(θ/2) + kc) (where k is a plotting constant) and extrapolating the data to zero angles

and concentrations, radius of gyration (Rg) and second viral coefficient (A2) can be

obtained from the slopes respectively. A simultaneous extrapolation to zero angle and

concentration yields an intercept, which is the inverse of Mw.

Scanning electron microscopy (SEM). The morphologies of the peptide nanoparticles

and microorganisms before and after treatment with peptide nanoparticles were observed

using a field emission SEM (JEOL JSM-7400F) operated at an accelerating voltage of

4.0-6.0 keV. For peptide nanoparticles, 20 μL of the nanoparticle solution was placed on

a silicon wafer, and air-dried at room temperature. The wafer was mounted on aluminum

stud, and then coated with platinum for visualization.

The microorganisms grown in broth alone or incubated with peptide nanoaprticles were

harvested by centrifugation at 2500g for 10 minutes. They were washed with phosphate-

buffered saline (PBS) for three times and then fixed in PBS containing 5% formaldehyde

for one day. The cells were further washed with DI water, followed by dehydration using

a series of ethanol washes and drying in a critical point dryer (Autosamdri-815, Tousimis

Research Corporation, U.S.A.). The cell sample was placed on carbon tape, which was

mounted onto aluminum stud, and coated with platinum prior to SEM analyses.

3




0.55

0.60

0.65

0.70

0.75

0.80

0.85

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5Log C

I339

/I334

Figure S1 Plot of I339/I334 ratio as a function of logarithm of peptide concentration (Log C, mg/L) in DI water.

Hydrodynamic radius, radius of gyration and aggregation numbers

Dynamic light scattering (DLS) and static light scattering (SLS) experiments were

performed on a Brookhaven BI-200SM goniometer system (U.S.A.) to determine the

hydrodynamic radius (Rh), gyration of radius (Rg) and weight average molecular weight

(Mw) respectively. The light source is a power adjustable and vertically polarized 75 mW

HeNe ion laser with a wavelength of 633 nm. For SLS, a Zimm plot (Eq 1) was used to

analyze the experimental data and the refractive index increment (dn/dc) was measured

using a BI DNDC differential refractometer (Brookhaven, U.S.A.). Samples are

equilibrated for 10 minutes before measurements were taken. The aggregation number

(Nw) was determined by the quotient of the micelle Mw and the unimer Mw.

( ) cARn

MRKc g

w22

2222

23

2sin16

11+

⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜

⎝

⎛⎟⎠⎞

⎜⎝⎛

+=λ

θπ

θ

2

where K (= ( ) 42224 λπ ANdcdnn ) is an optical constant, R(θ) (= θsin2ts IrI ) is the

Rayleigh ratio, c is the concentration of the polymer solution in gram per milliliter, n is

the refractive index of the solvent, θ is the angle of measurement, λ is the wavelength of

the laser in vacuum and NA is the Avogadro’s constant. A plot of (Kc/R(θ)) against

(sin2(θ/2) + kc) (where k is a plotting constant) and extrapolating the data to zero angles

and concentrations, radius of gyration (Rg) and second viral coefficient (A2) can be

obtained from the slopes respectively. A simultaneous extrapolation to zero angle and

concentration yields an intercept, which is the inverse of Mw.

Scanning electron microscopy (SEM). The morphologies of the peptide nanoparticles

and microorganisms before and after treatment with peptide nanoparticles were observed

using a field emission SEM (JEOL JSM-7400F) operated at an accelerating voltage of

4.0-6.0 keV. For peptide nanoparticles, 20 μL of the nanoparticle solution was placed on

a silicon wafer, and air-dried at room temperature. The wafer was mounted on aluminum

stud, and then coated with platinum for visualization.

The microorganisms grown in broth alone or incubated with peptide nanoaprticles were

harvested by centrifugation at 2500g for 10 minutes. They were washed with phosphate-

buffered saline (PBS) for three times and then fixed in PBS containing 5% formaldehyde

for one day. The cells were further washed with DI water, followed by dehydration using

a series of ethanol washes and drying in a critical point dryer (Autosamdri-815, Tousimis

Research Corporation, U.S.A.). The cell sample was placed on carbon tape, which was

mounted onto aluminum stud, and coated with platinum prior to SEM analyses.

3




Transmittance electron microscopy (TEM)

The morphologies of the microorganisms before and after treatment with peptide

nanoparticles were observed under a JEM-1230 transmittance electron microscope

(JEOL, Japan) using an acceleration voltage of 80 keV. The microorganism solutions (1.5

mL) were incubated with 0.5 mL of peptide nanoparticle solution (100 mg/L) for 2 hours.

The solutions were centrifuged at 5000 rpm for 10 minutes, and the supernatants were

removed. Phosphate buffer (pH 7.0, 1.5 mL) was mixed with the microorganisms, and

then centrifuged at 5000 rpm for 10 minutes to remove the phosphate buffer. 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 samples were

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

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

samples were washed three times in the phosphate buffer (15 minutes each),

followed by dehydration in a graded ethanol series.

The samples were 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.

Hemolysis assays. Fresh rat 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 peptide nanoparticle or amphotericin B solution was added to

4

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. The tests were repeated

eight times, and the data are expressed as mean and standard deviation of 8 replicates.

In vivo studies

Determination of minimum lethal dose of Staphylococcus aureus. Five groups of 6

mice each were inoculated intraperitoneally with 0.5 mL of Staphylococcus aureus

suspension containing various doses of Staphylococcus aureus (i.e. 1.0 × 109, 1.63 × 109,

2.66 × 109, 4.33 × 109 and 7.0 × 109 CFU/mL). Mortality was monitored at 48 hours post-

infection, which was 1, 2, 4, 6 and 5 respectively. The minimum lethal dose of

Staphylococcus aureus was determined through the method of BLISS.

ED50. Five groups of 6 mice each were inoculated intraperitoneally with 0.5 mL of

Staphylococcus aureus suspension at the minimum lethal dose (i.e. 4.34×109 CFU/mL).

Immediately after the infection, the mice received intravenous injection of peptide

nanoparticles at various doses (i.e. 0.45, 0.8, 1.4, 2.5 and 4.5 mg/kg, 0.1 mL), and another

injection with the nanoparticles at the same doses at 24 hours. Survival of mice was

5




Transmittance electron microscopy (TEM)

The morphologies of the microorganisms before and after treatment with peptide

nanoparticles were observed under a JEM-1230 transmittance electron microscope

(JEOL, Japan) using an acceleration voltage of 80 keV. The microorganism solutions (1.5

mL) were incubated with 0.5 mL of peptide nanoparticle solution (100 mg/L) for 2 hours.

The solutions were centrifuged at 5000 rpm for 10 minutes, and the supernatants were

removed. Phosphate buffer (pH 7.0, 1.5 mL) was mixed with the microorganisms, and

then centrifuged at 5000 rpm for 10 minutes to remove the phosphate buffer. 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 samples were

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

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

samples were washed three times in the phosphate buffer (15 minutes each),

followed by dehydration in a graded ethanol series.

The samples were 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.

Hemolysis assays. Fresh rat 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 peptide nanoparticle or amphotericin B solution was added to

4

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. The tests were repeated

eight times, and the data are expressed as mean and standard deviation of 8 replicates.

In vivo studies

Determination of minimum lethal dose of Staphylococcus aureus. Five groups of 6

mice each were inoculated intraperitoneally with 0.5 mL of Staphylococcus aureus

suspension containing various doses of Staphylococcus aureus (i.e. 1.0 × 109, 1.63 × 109,

2.66 × 109, 4.33 × 109 and 7.0 × 109 CFU/mL). Mortality was monitored at 48 hours post-

infection, which was 1, 2, 4, 6 and 5 respectively. The minimum lethal dose of

Staphylococcus aureus was determined through the method of BLISS.

ED50. Five groups of 6 mice each were inoculated intraperitoneally with 0.5 mL of

Staphylococcus aureus suspension at the minimum lethal dose (i.e. 4.34×109 CFU/mL).

Immediately after the infection, the mice received intravenous injection of peptide

nanoparticles at various doses (i.e. 0.45, 0.8, 1.4, 2.5 and 4.5 mg/kg, 0.1 mL), and another

injection with the nanoparticles at the same doses at 24 hours. Survival of mice was

5




monitored for 7 days post-infection, which was 3, 5, 4, 4 and 6 respectively, and ED50

was determined through the method of BLISS.

LD50. Five groups of 6 mice each received intravenous injection of peptide nanoparticles

at various doses (i.e. 14.0, 16.2, 18.7, 21.7 and 25.0 mg/kg, 0.1 mL). Mortality was

monitored for 2 days post-treatment, which was 0, 2, 4, 6 and 6 respectively, and LD50

was determined using the method of BLISS.

Determination of percentage of neutrophils. The pathogen-free New Zealand rabbits

(approximately 2.5 to 3 kg) received 1.75 g/kg ketamine intramuscularly and then 10

mg/kg diazepam intravenously to induce deep anaesthesia. A spinal needle was inserted

into the cisterna magna, and 0.5 mL of CSF was taken. The bacterial suspension (2.5×106

CFU in 0.5 mL of saline solution) was injected into the subarachnoid space. After

inoculation, the animals were brought back to the cages overnight. After 12 hours of

incubation, the rabbits were separated into three groups (six rabbits for each group):

control, vancomycin therapy (20 mg/kg) and peptide nanoparticles therapy (0.35 mg/kg).

Antibiotics were given intravenously every 12 hours. CSF and blood samples were

obtained at different time points via the intracisternal puncture and auricular artery

respectively. Percentage of neutrophils in the peripheral blood was analyzed by a Coulter

Micro Diff Auto-hemocyte analyzer (U.S.A.). Percentage of neutrophils in the CSF was

analyzed through differential cell count with a light microscope (Olympus, Japan). The

CSF was centrifuged at 1,000 g for 10 minutes at 4°C to remove the supernatant, and

stained with Wright. The data are expressed as mean and standard deviation of 6 values

obtained from six rabbits.

6




monitored for 7 days post-infection, which was 3, 5, 4, 4 and 6 respectively, and ED50

was determined through the method of BLISS.

LD50. Five groups of 6 mice each received intravenous injection of peptide nanoparticles

at various doses (i.e. 14.0, 16.2, 18.7, 21.7 and 25.0 mg/kg, 0.1 mL). Mortality was

monitored for 2 days post-treatment, which was 0, 2, 4, 6 and 6 respectively, and LD50

was determined using the method of BLISS.

Determination of percentage of neutrophils. The pathogen-free New Zealand rabbits

(approximately 2.5 to 3 kg) received 1.75 g/kg ketamine intramuscularly and then 10

mg/kg diazepam intravenously to induce deep anaesthesia. A spinal needle was inserted

into the cisterna magna, and 0.5 mL of CSF was taken. The bacterial suspension (2.5×106

CFU in 0.5 mL of saline solution) was injected into the subarachnoid space. After

inoculation, the animals were brought back to the cages overnight. After 12 hours of

incubation, the rabbits were separated into three groups (six rabbits for each group):

control, vancomycin therapy (20 mg/kg) and peptide nanoparticles therapy (0.35 mg/kg).

Antibiotics were given intravenously every 12 hours. CSF and blood samples were

obtained at different time points via the intracisternal puncture and auricular artery

respectively. Percentage of neutrophils in the peripheral blood was analyzed by a Coulter

Micro Diff Auto-hemocyte analyzer (U.S.A.). Percentage of neutrophils in the CSF was

analyzed through differential cell count with a light microscope (Olympus, Japan). The

CSF was centrifuged at 1,000 g for 10 minutes at 4°C to remove the supernatant, and

stained with Wright. The data are expressed as mean and standard deviation of 6 values

obtained from six rabbits.

6

Bacterial counts in brain tissues. The brain tissue samples were weighed and placed in

a sterile glass homogenizer containing 1 mL of isotonic sodium chloride solution, which

were then homogenized. The homogenate (200 μL) was placed on a MH agar plate

(bioMérieux), and incubated overnight at 37°C in 5% CO2. The number of colony-

forming unit (CFU) was counted and expressed as lg CFU/g of tissues. The data are

expressed as mean and standard deviation of 6 values obtained from six rabbits.

7




0.00.10.20.30.40.50.60.70.80.91.0

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Control124.18.111.41316.2

µM µM µM

µM µM µM

µM

a

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c

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d

e

f

9




0.00.10.20.30.40.50.60.70.80.91.0

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µM µM µM

µM µM µM

µM

a

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µM µM µM µM µM

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c

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d

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0.0

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0.0

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h

µM µM µM µM µM µM µM

Figure S2 Dose-dependent growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA) (a, MIC: 11.4 µM), Candida albicans (b, MIC: 10.8 µM), Enterococcus faecalis (c, MIC: 11.4 µM), vancomycin-resistant Enterococcus (d, MIC: 4.1 µM), Cryptococcus neoformans (e, MIC: 8.1 µM), Candida tropicalis (f, MIC: 13.0 µM), Candida albicans 1 (g, MIC: 13.0 µM) and Streptococcus hemolyticus (h, MIC: 2.0 µM), in the presence of peptide nanoparticles.

10




0.0

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. at 6

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h

µM µM µM µM µM µM µM

Figure S2 Dose-dependent growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA) (a, MIC: 11.4 µM), Candida albicans (b, MIC: 10.8 µM), Enterococcus faecalis (c, MIC: 11.4 µM), vancomycin-resistant Enterococcus (d, MIC: 4.1 µM), Cryptococcus neoformans (e, MIC: 8.1 µM), Candida tropicalis (f, MIC: 13.0 µM), Candida albicans 1 (g, MIC: 13.0 µM) and Streptococcus hemolyticus (h, MIC: 2.0 µM), in the presence of peptide nanoparticles.

10

0

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a µM

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Control57.8115.6173.4231.2289

Figure S3 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: 290.0 µM) and Candida albicans (b, MIC: 289.0 µM) in the presence of G3TAT.

µMµM

µMµMµM

µM µM µM

µM µM µM µM

b

11




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Figure S4 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: 75.0 µM) and Candida albicans (b, MIC: 75.0 µM) in the presence of G3R6TAT.

b

µM µM

µM

µM µM

12

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a

µM µM µM

µM µM µM µM µM

bµMµMµMµMµM

Figure S5 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: 242.0 µM) and Candida albicans (b, MIC: 145.5 µM) in the presence of G3R12.

13




0.0

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Figure S4 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: 75.0 µM) and Candida albicans (b, MIC: 75.0 µM) in the presence of G3R6TAT.

b

µM µM

µM

µM µM

12

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. at 6

00 n

m

Control48.597145.5194242

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 15Time (hours)

O.D

. at 6

00 n

m

control 4.89.712.124.248.597121242

a

µM µM µM

µM µM µM µM µM

bµMµMµMµMµM

Figure S5 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: 242.0 µM) and Candida albicans (b, MIC: 145.5 µM) in the presence of G3R12.

13




0.0

0.1

0.2

0.3

0.4

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0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 15Time (hours)

O.D

. at 6

00 n

m

Control 8.917.822.244.488.9177.8222.2444.4

a

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6 8 10 16Time (hours)

O.D

. at 6

00 n

m

Control88.9177.8266.7355.6444.4 µM

µM µM µM µM

b

Figure S6 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: > 444.4 µM) and Candida albicans (b, MIC: > 444.4 µM) in the presence of G3R6.

µM µM

µM µM µM

µM

µM µM

14

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.11 0.22 0.43 0.85 1.7 3.4 6.8 13.5 27

O.D

. at 6

00 n

m

Figure S7 Dose-dependent growth inhibition of Bacillus subtilis in the presence of penicillin G (a, MIC: 1074 µM) and doxycycline (b, MIC: 13.5 µM) (16 hours of incubation).

Drug concentration (µM)

b

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

8.3 16.8 33.6 67.1 134 209 419 838 1074 1342

O.D

. at 6

00 n

m


a

15




0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 1 2 3 4 5 6 7 8 15Time (hours)

O.D

. at 6

00 n

m

Control 8.917.822.244.488.9177.8222.2444.4

a

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6 8 10 16Time (hours)

O.D

. at 6

00 n

m

Control88.9177.8266.7355.6444.4 µM

µM µM µM µM

b

Figure S6 Dose-dependent growth inhibition of Bacillus subtilis (a, MIC: > 444.4 µM) and Candida albicans (b, MIC: > 444.4 µM) in the presence of G3R6.

µM µM

µM µM µM

µM

µM µM

14

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.11 0.22 0.43 0.85 1.7 3.4 6.8 13.5 27

O.D

. at 6

00 n

m

Figure S7 Dose-dependent growth inhibition of Bacillus subtilis in the presence of penicillin G (a, MIC: 1074 µM) and doxycycline (b, MIC: 13.5 µM) (16 hours of incubation).


b

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

8.3 16.8 33.6 67.1 134 209 419 838 1074 1342

O.D

. at 6

00 n

m


a

15




G3R6TAT

CG3R6TAT

Figure S8 MALDI-TOF mass spectra of G3R6TAT and CG3R6TAT. The spectrum of G3R6TAT shows its theoretical molecular weight at 2667 Da, indicating the successful synthesis of the peptide. The theoretical molecular weight of CG3R6TAT was 3080 Da, which appears in the spectrum of CG3R6TAT, indicating successful conjugation of cholesterol.

16

G3R6TAT

CG3R6TAT

a

b

Figure S9 1H-NMR spectra of CG3R6TAT and G3R6TAT in d-DMSO. The weak and ultiple peaks at δ 0.7-1.1 (signal a) were from cholesterol. The multiple peaks at δ 6.7-8.5

signal b) were attributed to the protons from the benzene ring of tyrosine in the peptide. hese findings further prove successful conjugation of cholesterol onto the peptide.

m(T

17




G3R6TAT

CG3R6TAT

Figure S8 MALDI-TOF mass spectra of G3R6TAT and CG3R6TAT. The spectrum of G3R6TAT shows its theoretical molecular weight at 2667 Da, indicating the successful synthesis of the peptide. The theoretical molecular weight of CG3R6TAT was 3080 Da, which appears in the spectrum of CG3R6TAT, indicating successful conjugation of cholesterol.

16

G3R6TAT

CG3R6TAT

a

b

Figure S9 1H-NMR spectra of CG3R6TAT and G3R6TAT in d-DMSO. The weak and ultiple peaks at δ 0.7-1.1 (signal a) were from cholesterol. The multiple peaks at δ 6.7-8.5

signal b) were attributed to the protons from the benzene ring of tyrosine in the peptide. hese findings further prove successful conjugation of cholesterol onto the peptide.

m(T

17




Table S1 Effect of oligopeptide nanoparticles on the liver and kidney functions

Treatment ALT (U/L) AST (U/L) Total bilirubin (μmol/L)

Direct bilirubin (μmol/L)

Creatinine (μmol/L)

Urea nitrogen (mmol/L)

w/o treatment 100.2±45.9

(n=6)

45.0±21.4

(n=6)

2.0±0.0

(n=6)

1 ∼ 7

(n=6)

73.2±3.9

(n=6)

6.6±1.1

(n=6)

with the

nanoparticles

treatment

112.7±12.0

(n=6)

p=0.6 > 0.05

20.5±6.8

(n=6)

p = 0.05

1.2±0.4

(n=6)

p < 0.05

1 ∼ 7

(n=6)

54.2±6.2

(n=6)

p < 0.05

7.2±1.6

(n=6)

p=0.3> 0.05

18


Documents

SUPPLEMENTARY INFORMATION Supplementary Information … · nm) were cut using a Reichert-Jung Ultracut E ultramicrotome, and post-stained with uranyl acetate and lead citrate for