electronic supplementary information measurements 1h nmr

Electronic Supplementary Information

Measurements

1H NMR spectra were recorded at 300 MHz on a Mercury VX-300 spectrometer by using

tetramethylsilane (TMS) as the internal reference. 13C-NMR spectra were recorded at 75 MHz

with the solvent carbon signal as reference. Infrared spectra were recorded on a Nicolet Avator

360 FT-IR spectrometer. Size and distribution measurements of the micelles were performed on a

Nano-ZSZEN3600 (Malvern) instrument at room temperature. The critical micelle concentration

(CMC) was determined by using pyrene as a fluorescence probe. The concentration of polymer

was varied from approx. 1.0 × 10−3 to 0.5 mg/mL and the concentration of pyrene was fixed at 6 ×

10−7

The HABA/avidin assay was performed according to an early report described by Wooly.

M. The fluorescence spectra were recorded using an FLS920 fluorescence spectrometer with

an excitation wavelength of 337 nm. The emission fluorescence at 373 and 383 nm was monitored.

The CMC was estimated as the intersection when extrapolating the intensity ratio I373/I383 at low

and high concentration regions. Transmission electron microscopy images (TEM) were obtained

using a JEM-100CXⅡ transmission electron microscope. A drop of micelle solution (0.1 mg/mL)

was placed onto a copper grid with carbon film. The TEM images was observed at an acceleration

voltage of 80 keV.

[1] The

HABA solution was prepared by dissolving 24.2 mg of 4-hydroxyazobenzene-2-carboxylic acid

(0.1 mmol) in 10 mL of aqueous sodium hydroxide solution (10 mM). Then the HABA/Avidin

solution was made by dissolving 5.0 mg of avidin in 9.7 mL of 50 mM PBS with 50mM NaCl

(pH=7.2), followed by adding HABA solution (300 μL). This solution was stored up to one week

at 4ºC. The CPCB micelle stocks were prepared in different PBS solution (0.1 M, pH=6.8 or 7.4)

and kept in 37ºC for 4 h. The analyses were performed after mixing the HABA/Avidin solution

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2012

(300 μL) with the samples (33.3 μL). The final concentration of micelle was set at 0.1 mg/mL.

The UV spectra were recorded on a UV/Vis microplate spectrophotometer (Multiskan GO,

Thermo Fisher). The amount of biotin moieties available to avidin in the samples was calculated

by the formula: (ΔA500/34)×1000. (ΔA500

Cell internalization was observed on a confocal laser scanning microscopy (CLSM, Nikon C1-si).

MCF-7 cells were seeded in a 6-well plate and incubated at 37°C for 24 h. The mixed micelles

(CPCB/CPD/CPFITC= 0.1/0.1/0.01, CPC/CPD/CPFITC= 0.1/0.1/0.01) in fresh prepared DMEM

with 10 % fetal bovine serum (FBS) at different pH values were added to replace the medium.

After 4 h of incubation, the medium was removed and the cells were washed 3 times with PBS.

Then the cells were fixed with 4 wt % formaldehyde in PBS for 10 min at room temperature. A

drop of mounting media (10% PBS, 90% glycerol) were added to mount the cells. The

fluorescence of dox and fitc was examined under excitation at 488 nm.

means the differences of the UV absorbance at 500 nm

before and after the addition of the micelle samples).

The release profiles of DOX from nano-flowers were studied at 37°C under three different pH

conditions, i.e. (a) acetate buffer, pH 5.4; (b) phosphate buffer, pH 6.8; and (c) phosphate buffer,

pH 7.4. All the concentrations of the release media were 0.1 M. Every 1 mL of the mixed-micelles

solution was transferred to a dialysis tube (Mw 3500, cutoff). Then dialysis tube was immersed

into 20 mL of the corresponding buffer and the medium was shaken at 37°C. At desired time

intervals, 2 mL of release medium was replaced with an equal volume of fresh medium and then

dialyzed. The amount of released DOX was calculated by the results recorded on FLS920

fluorescence spectrometer. All the release experiments were conducted in triplicate. The results

presented are the average value ± standard deviation.


The cytotoxicity assessment was carried out in MCF-7 cells by using the MTT assay.

100 μL of cell suspension (DMEM with 10% FBS) containing 6×103 MCF-7 cells

were seeded into each well of a 96-well plate and incubated at 37°C with 5% CO2

Cell viability (%) = (A

for

24 h. Then the cells were treated with mixed-micelles at various concentrations and

carried out a further incubation for 24 h. After that, all mediums were replaced with

MTT reagent (20 μL in PBS, 5 mg/mL) and incubated for another 4 h. The medium in

each well was carefully removed and replaced by 100 μL DMSO. When the purple

solution was homogeneous, the absorbance at 570 nm was recorded by a microplate

reader (Multiskan GO, Thermo Fisher). Cell viability was calculated by

treated/Acontrol

The data are shown as the average value ± standard deviation.

) ×100%

Materials: D-Biotin, 1,1'-carbonyldiimidazole (CDI), di-tert-butyl dicarbonate ((Boc)2O),

hydrazine hydrate,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl),

sodium azide, 3-chloropropylamine hydrochloride, 1-octadecanol, 4-(dimethylamino)-pyridin

(DMAP), dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were purchased

from Sinopharm (China) and used as received. N,N,N',N'',N''-pentamethyldiethylenetriamine

(PMDETA) , propargylamine, 4’-hydroxyazobenzene-2-carboxylic acid (HABA) and Fluorescein

isothiocyanate isomer I (FITC) were purchased from Alfa aesar and used as received. Doxorubicin

hydrochloride (Dox · HCl) was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (China).

Trifluoroacetic acid, bromoacetyl bromide and 4-formylbenzoic acid were purchased from

Aladdin-reagent (China) and used as received. CuBr, Brij S 100 (average Mn ~4670) and


H-lys(Boc)-OH were purchased from Sigma-Aldrich and used as received. N,N-dimethyl

formamide (DMF) was dried over 4 Å molecular sieve and distilled over anhydrous MgSO4.

Dichloromethane (DCM) was distilled over CaH2

t-butyl carbazate 1

before used. Methanol was dried over 3 Å

molecular sieve and distilled by rectification. Bovine serum albumin (BSA), Dubelcco’s Modified

Eagle’s Medium (DMEM), penicillin–streptomycin, trypsin, and phosphate-buffered saline (PBS),

and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from

GIBCO Invitrogen Corporation.

Hydrazine hydrate (5.0 g, 0.1 mol) was dissolved in THF (30 mL) at 0ºC, and treated dropwise

with a solution of Boc2O (10.0 g, 45.8 mmol) in 10 mL of THF. Then the reaction was kept

stirring at room temperature over night. The solvent was removed and the residue was dissolved in

DCM, washed with distilled water and dried over anhydrous Na2SO4. The DCM was evaporated

and the residue was distilled under reduced pressure to obtain t-butyl carbazate as a white solid.

Yield: 4.5 g, (74.4 %). 1H NMR (300 MHz, CDCl3, δ ppm): 1.42 (s, 9H), 3.68 (s, 2H), 6.13 (s,

1H). 13C NMR (75 MHz, CDCl3

N-boc-2-bromoacetohydrazide 2

, δ ppm): 28.5, 77.2, 158.3.

t-Butyl carbazate (2.0 g, 15.2 mmol) and TEA (1.6 g, 15.8 mmol) were stirred in DCM (20 mL) at

0°C, and treated dropwise with a solution of bromoacetyl bromide (3.1 g, 15.4 mmol) in 1 mL of

DCM. The mixture was kept stirring at room temperature over night and filtrated, washed with

saturated NaHCO3 and brine, dried over anhydrous Na2SO4. The solvent was removed by rotary

evaporation and the residue was passed through a silica column (eluent, hexane/ethyl acetate =

1/1). N-boc-2-bromoacetohydrazide was obtained as colorless solid. Yield: 3.3 g (74.4 %). 1H


NMR (300MHz, CDCl3 δ ppm): 1.48 (s, 9H), 3.92(s, 2H), 7.14 (m, 1H), 8.92 (m, 1H). 13C NMR

(75 MHz, CDCl3

N-boc-2-azideacetohydrazide 3

, δ ppm): 26.9, 28.8, 83.1, 156.2, 166.7.

N-boc-2-bromoacetohydrazide (2.0 g, 7.9 mmol) and sodium azide (3.2g, 32 mmol) were

dissolved in 20 mL of anhydrous DMF and was stirred for 24h at 40°C. Then the solvent was

removed by rotary evaporation and the residue was dissolved in ether, washed with distilled water

for three times and dried over anhydrous Na2SO4. The solvent was removed and the product was

obtained as colorless solid. Yield: 1.6g (94.2%). 1H NMR (300MHz, CDCl3, δ ppm): 1.48 (s, 9H),

4.06(s, 2H), 6.97(m, 1H), 8.55 (m, 1H). 13C NMR (75 MHz, CDCl3

DOX-N

, δ ppm): 28.8, 51.9, 83.1,

156.1, 167.5.

3

N-boc-2-azideacetohydrazide (0.08g, 0.37 mmol) was stirred in a mixture solution of DCM and

TFA (1/1) for 30 min at room temperature. Then the solvent was removed by rotary evaporation

and the residue was co-evaporated three times with toluene to remove the excess TFA. The

obtained yellowish oil was dried over P

4

2O5 and NaOH under vacuum. The above yellowish oil

and Dox HCl (0.2g, 0.34 mmol) were dissolved in 15mL of anhydrous menthol, and treated with a

drop of TFA. After refluxed for 48h under dark, the mixture was cooled down. The solvent was

removed by rotary evaporation and the residue was precipitated in excess ethyl acetate. Precipitate

was collected by centrifugation and washed with ether. The crude product was obtained as a dark

red solid. DOX-N3

Aminopropylazide 5

was used in the next steps without further purification. Yeild: 97 mg (41.1%).

3-Chloropropylamine hydrochloride (10.0g, 78.0 mmol) and sodium azide (15.0g, 231.0 mmol)


and potassium iodide (1.3g, 7.8 mmol) were dissolved in 150 mL of distilled water and heated at

80°C for 18 h. The reaction solution was concentrated under vaccum and the pH was adjusted to

10 by adding aqueous NaOH solution (4M). The aqueous phase was extracted using diethyl ether,

and then the organic phase was dried over anhydrous Na2SO4, filtrated and concentrated. The

product was purified by distillation under vaccum and dried over molecular sieve. Yield: 5.3 g

(67.9 %).1H NMR (300 MHz, CDCl3, δ ppm): 1.14 (bs, 2H), 1.74 (m, 2H), 2.81 (t, 2H), 3.38 (t,

2H). 13C NMR (75 MHz, CDCl3

Octadecyl 4-formylbenzoate 6

, δ ppm): 32.1, 39.1, 49.1.

4-Formylbenzoic acid (3.0 g, 20.0 mmol), 1-octadecanol (5.4 g, 20.0 mmol), DMAP (0.24 g, 2.0

mmol) and DCC (4.1 g, 20.0 mmol) were dissolved in anhydrate ethyl ether (150 mL) and the

mixture was allowed to stir at room temperature for 24 h. Then the suspension was filtrated and

concentrated. The residue was purified by column chromatography (silica, EtOAc/petroleum ether

= 4:1 v/v). Octadecyl 4-formylbenzoate was obtained as a white solid. Yield: 5.8 g, (72.1 %). 1H

NMR (300MHz, CDCl3, δ ppm): 0.88 (t, 3H), 1.26 (m, 30H), 1.79 (m, 2H), 4.35 (t, 2H), 7.96 (d,

2H), 8.21 (d, 2H), 10.10 (s, 1H). 13C NMR (75 MHz, CDCl3

Octadecyl 4-((prop-2-ynylimino) methyl) benzoate 7

, δ ppm): 22.9, 26.3, 28.9, 29.5, 29.6,

29.9, 32.2, 66.0, 129.7, 130.4, 135.7, 139.3, 165.8, 191.8.

Octadecyl 4-formylbenzoate (2.0 g, 5.0 mmol) and propargylamine (0.28 g, 5.0 mmol) were

dissolved in anhydrous methanol (30 mL) and the solution was refluxed for 30 min. Then the hot

solution was cooled down to room temperature and recrystallization was carried out at 4ºC for a

further 10 h. The production was collected by filtration and then washed by methanol. Octadecyl

4-((prop-2-ynylimino) methyl) benzoate was obtained as a yellow solid. Yield: 1.8 g, (82.0 %). 1H


NMR (300MHz, CDCl3, δ ppm): 0.88 (t, 3H), 1.25 (m, 30H), 1.78 (m, 2H), 2.56 (s, 1H), 4.33 (t,

2H), 4.58 (s, 2H), 7.85 (d, 2H), 8.11 (d, 2H), 8.65 (s, 1H). 13C NMR (75 MHz, CDCl3

Octadecyl 4-((3-azidopropylimino) methyl) benzoate 8

, δ ppm):

22.9, 26.3, 28.9, 29.5, 29.6, 29.7, 29.8, 29.9, 32.2, 47.4, 65.6, 76.3, 78.7, 128.3, 130.0, 132.7,

139.8, 161.5, 166.3.

Octadecyl 4-formylbenzoate (2.0 g, 5.0 mmol) and aminopropylazide (0.5 g, 5mmol) were

dissolved in anhydrous methanol (30 mL) and the solution was refluxed for 30 min. Then the hot

solution was cooled down to room temperature and recrystallization was carried out at 4°C for a

further 10 h. The production was collected by filtration and then washed by methanol. Octadecyl

4-((3-azidopropylimino) methyl) benzoate was obtained as a white solid. Yield: 2.1 g (86.8 %). 1H

NMR (300MHz, CDCl3, δ ppm): 0.88 (t, 3H), 1.25 (m, 30H), 1.77 (m, 2H), 2.02 (m, 2H), 3.42 (t,

2H), 3.73 (t, 2H), 4.32 (t, 2H), 7.80 (d, 2H), 8.10 (d, 2H), 8.36 (s, 1H). 13C NMR (75 MHz, CDCl3

Biotin-NHS 9

,

δ ppm): 22.9, 26.3, 28.9, 29.5, 29.6, 29.7, 29.8, 29.9, 30.2, 32.2, 49.4, 58.5, 65.6, 128.2, 130.1,

132.7, 139.8, 161.2, 166.3.

Biotin (5 g, 20.5 mmol) was dissolved in 150 mL of DMF at 60°C, and then treated with DCC

(4.3 g, 20.8 mmol) and NHS (2.5 g, 21.7 mmol). The mixture was stirred at 60°C for 2 h and kept

stirring for 24 h at room temperature. The precipitate formed during reaction was filtrated and the

volume of solvent was concentrated to 30 mL by rotary evaporation, and then excess acetone was

added into the mixture until no more precipitate formed. The precipitate was collected and washed

with acetone. Biotin-NHS was purified by recrystallization in isopropanol. Yield: 4.3 g,

(61.5 %). 1H NMR (300MHz, DMSO-d6, δ ppm): 1.25~1.68 (m, 6H), 2.55 (d, 1H), 2.67 (t, 2H),


2.80~2.86 (m, 5H), 3.10 (t, 1H), 4.14 (m, 1H), 4.30 (m, 1H) 6.37 (d, 1H), 6.44 (d, 1H). 13C NMR

(75 MHz, DMSO-d6

Biotin-lys(Boc)-OH 10

, δ ppm): 24.7, 25.9, 28.0, 28.3, 30.4, 55.6, 59.86, 61.4, 163.1, 169.4, 170.7.

H-lys(Boc)-OH (1.5 g, 6.1 mmol)was suspended in 100 mL of DMF, and treated with biotin-NHS

(2.0 g, 5.8 mmol) and TEA (2.4 g, 11.8 mmol). The mixture was stirred at room temperature until

most of solid were dissolved (this process needed 2 days at least) and another TEA (2.4 g, 11.8

mmol) was added. After the solution sirred for 48 h at room temperature, the small amount of

insoluble substances were filtrated and the solvent was removed by rotary evaporation. The

residue was dissolved in n-butanol and then washed three times with 0.5 M KHSO4, three times

with distilled water, once with brine and dried over hydrous Na2SO4. The biotin-lys(Boc)-OH was

dried over P2O5 and NaOH under vacuum and was obtained as a yellowish solid. Yield: 2.1 g

(76.7 %). 1H NMR (300MHz, DMSO-d6, δ ppm): 1.18~1.72 (m, 21H), 2.11 (t, 2H), 2.56 (d, 1H),

2.81~2.86 (m, 3H), 3.09 (t, 1H), 4.11~4.14 (m, 2H), 4.30 (m, 1H), 6.35 (d, 1H), 6.41 (d, 1H), 6.74

(t, 1H), 7.87~8.00 (m, 1H). 13C NMR (75 MHz, DMSO-d6

Biotin-lys(Boc)-N

, δ ppm): 23.3, 25.7, 28.7, 29.6, 31.2,

33.8, 35.2, 36.2, 52.2, 55.9, 59.7, 61.5, 77.8, 156.0, 163.2, 172.7, 174.3.

3

Biotin-lys(Boc)-OH (2g, 4.2 mmol) and EDC·HCl (0.81g, 4.2 mmol) were dissolved in 100 mL of

DMF and then aminopropylazide (0.5g, 5.0 mmol) was added. After the solution was stirred for

24h at room temperature, the solvent was removed by rotary evaporation. The residue was

dissolved in n-butanol and washed once with 0.5 M KHSO

11

4, three times with distilled water, once

with brine, dried over hydrous Na2SO4 and filtrated. The filtrate was concentrated and added into

excess ethyl ether for precipitation. The precipitate was collected and purified with column


chromatography (silica, DCM/MeOH=10:1 v/v). Biotin-lys(Boc)-N3 was obtained as a yellowish

solid. Yield: 1.9g (81.6 %). 1H NMR (300MHz, DMSO-d6, δ ppm): 1.18~1.72 (m, 23H), 2.11 (t,

2H), 2.56 (d, 1H), 2.78~2.87 (m, 3H), 3.09 (m, 3H), 3.32~3.37 (m, 2H), 4.14 (m, 2H), 4.30 (m,

1H), 6.39 (d, 1H), 6.47 (d, 1H), 6.73 (t, 1H), 7.87~7.98 (m, 2H). 13C NMR (75 MHz, DMSO-d6

CDI-activated PEG-b-C18 12

, δ

ppm): 23.3, 25.6, 28.4, 28.5, 28.7, 29.6, 32.2, 35.3, 36.2, 48.7, 52.9, 55.9, 59.7, 61.4, 77.7, 156.0,

163.2, 172.4, 172.5.

Brij (100) (6.1g, 1.3 mmol) was dissolved in DCM (60 mL) and CDI (2.07 g, 12.8 mmol) was

added, and then the solution was allowed to stir for 24h at room temperature. The reaction mixture

was concentrated to a small volume and poured into excess anhydrous diethyl ether. The white

precipitate was collected by centrifugation and washed with diethyl ether and dried under vacuum.

The product with trace imidazole was directly used in the next step without further purification.

Yield5.4 g, (87.2%). 1H NMR (300MHz, CDCl3,

Biotin-lys(PEG-C18)-N

δ ppm): 0.88 (t, 3H), 1.25 (m, 30H), 1.57 (m,

2H), 3.4~3.9 (m, ~400H), 4.56 (t, 2H), 7.08 (d, 1H), 7.46 (d, 1H), 8.16 (s, 1H).

3

Biotin-lys(Boc)-N

13

3 (0.55g, 1.0 mmol) was stirred in a mixture solution of DCM and TFA (1/9) for

2 h at room temperature. The solvent was then removed by rotary evaporation and the residue was

added into excess ethyl ether. The precipitate was collected and dried over P2O5 and NaOH under

vaccum for two days. The de-protected Biotin-lys(Boc)-N3 and PEG-b-C18-CDI (1.0g, 0.2 mmol)

were dissolved in hydrous DMF, and treated with TEA (0.21g, 2.1 mmol) to neutralize the trace

mount of TFA and catalyze the reaction. The mixture was stirred for 72 h at room temperature and

dialyzed against with DMSO for 72h (1L×6) and then dialyzed extensively against with DI water.


Biotin-lys(PEG-C18)-N3 was obtained by freeze-drying. Yield: 0.72g (69.9 %). 1H NMR

(300MHz, CDCl3

Alkyne-PEG-b-C18 14

, δ ppm): 0.88 (t, 3H), 1.25 (m, 34H), 1.57 (m, 12H), 3.4~3.9 (m, ~400H),

4.00~4.30 (m, 5H), 6.39 (d, 1H), 6.47 (d, 1H).

CDI-activated PEG-b-C18 (2g, 0.42 mmol), propargylamine (0.23g 4.2mmol) and triethylamine

(0.61mL, 4.2mmol) were dissolved in DCM (15mL) and the solution was allowed to stir for 48 h

at room temperature. The procedure of purification was same as that of compound 12. Yield: 1.6g

(80.2 %). 1H NMR (300MHz, CDCl3

Click chemistry

, δ ppm): 0.88 (t, 3H), 1.25 (m, 30H), 1.57 (m, 2H), 2.25 (s,

1h), 3.4~3.9 (m, ~400H), 4.15~4.31 (m, 4H).

C18-PEG-(biotin)-C18, C18-PEG-C18 and C18-PEG-DOX were synthesized in a similar

procedure. Typically, C18-PEG-(biotin)-N3 and octadecyl 4-((prop-2-ynylimino)methyl)benzoate

were dissolved in 2mL of DMF. After three freeze-pump-thaw cycles, PMDETA and Cu(Ⅰ)Br were

added and the solution was stirred under Ar atmosphere at 35°C for 24 h. After reaction, the

mixture was diluted with THF and passing through a short alumina column to eliminate the copper.

The product was obtained by three times of precipitation in excess ice-cooled ethyl ether and dried

under vacuum.

C18-PEG-DOX was synthesized in a similar procedure but purified by dialysis (DMSO for 72h

(1L×6), and then DI water for 24h (1L×4)).


Scheme S1. The synthesis route of CPCB. (g) EDC, aminopropylazide, in DMF, rt. 24 h; (h) 90% TFA, rt. 2 h; (i) TEA, in CH2Cl2, rt. 48 h; (l) propargylamine, in anhydrous CH3OH, reflux, 30 min; (m) CuBr, PMDETA, in DMF, three freeze-pump-thaw cycles, 35ºC, 24 h.


Scheme S2. The synthesis route of CPD. (a) bromoacetyl bromide, TEA, ice cool to rt. 24 h; (b) NaN3, DMF, 40°C, 24 h; (c) 50% TFA in CH2Cl2, rt. 4 h; (d) Dox•HCl, catalytic amount TFA, reflux in anhydrous CH3OH, 48 h in dark; (e) propargylamine and TEA, in CH2Cl2

, rt. 48 h; (f) CuBr, PMDETA, in DMF, three freeze-pump-thaw cycles, 35°C, 24 h, dialysis (DMSO for 72 h (1L×6), and then DI water for 24 h (1L×4)).


Figure S1. 1

H NMR spectrum of CPCB.


Figure S2. 1

H NMR spectrum of CPD.


Figure S3. 1

H NMR spectrum of CPC.


Figure S4. Change of FT-IR spectra in the synthesis of CPCB


Figure S5. Enlargement of Figure S4.


Figure S6. Change of FT-IR spectra in the synthesis of CPD.


Figure S7. Enlargement of Figure S6.


Figure S8. Determination of the critical micelle concentration of CPCB.


Figure S9. Determination of the critical micelle concentration of CPD.


Figure S10. Size distribution of CPCB measured by DLS.


Figure S11. Size distribution of CPD measured by DLS.


Figure S12. Confocal images of MCF-7 cells treated with CPCB/CPD mixed micelles and CPC/CPD mixed micelles.


electronic supplementary information measurements 1h nmr

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